Process for producing rfrp

ABSTRACT

The present invention is intended to provide an advantageous method of industrially producing RFamide peptides in a large scale. The present invention provides the method of producing RFamide peptides, which comprises subjecting a fusion protein or peptide, in which an RFamide peptide is ligated to the N-terminal of a protein or peptide having a cysteine at the N-terminal, to the reaction for cleavage of a peptide bond on the amino acid side of the cysteine residue.

TECHNICAL FIELD

[0001] The present invention relates to a method of producing a partialpeptide of a polypeptide comprising the amino acid sequence representedby SEQ ID NO: 19, NO: 21, NO: 23, NO: 25, NO: 27 or NO: 29, or a saltthereof, which comprises producing a fusion protein or polypeptide, andthen subjecting said fusion protein or polypeptide to a reaction ofcleaving a peptide bond. Further, the present invention relates to amethod of efficiently eliminating the N-terminal Met residue, optionallyoxidized, or a diketone of the Met residue from a partial peptide of apolypeptide comprising the amino acid sequence represented by SEQ ID NO:19, NO: 21, NO: 23, NO: 25, NO: 27 or NO: 29 having a Met residue at theN-terminal, optionally oxidized, or a salt thereof, if desired.

BACKGROUND ART

[0002] In the production of a peptide by recombinant DNA technology, itis more or less common practice to express the peptide in the form of afusion protein because of frequent decomposition of the peptide withincells. For excision of the target peptide from the fusion protein, achemical cleavage using cyanogen bromide (Itakura et al., Science, 198,1056, 1977) and an enzymatic cleavage using factor Xa (Nagai et al.,Methods in Enzymology, 153, 46, 1987) are known.

[0003] Further, as a method for cleavage of a peptide bond in a protein,cleavage of the acylcysteine bond with 2-nitro-5-thiocyanobenzoic acidis known (“Seikagaku Jikken Koza 1, Tanpakushitsu-no-Kagaku II(Biochemical Experiment Series 1, Protein Chemistry II)”, JapaneseSociety of Biochemistry ed., Tokyo Kagaku Dojin, 247-250, 1976).However, there is no disclosure on the excision of a partial peptide ofa polypeptide comprising the amino acid sequence represented by SEQ IDNO: 19, NO: 21, NO: 23, NO: 25, NO: 27 or NO: 29 from a protein.

[0004] The prior art method using cyanogen bromide cannot be applied tothe production of methionine-containing peptides, and has drawbacks, forexample in terms of excision yield.

[0005] Therefore, there is a need for a method of efficiently excising apartial peptide of a polypeptide comprising the amino acid sequencerepresented by SEQ ID NO: 19, NO: 21, NO: 23, NO: 25, NO: 27 or NO: 29from a fusion protein or polypeptide.

[0006] In addition, there may be differences in higher structure,biological activity, and stability between the molecular type containingMet at the N-terminal and the one not containing Met at the N-terminalof a partial peptide of a polypeptide comprising the amino acid sequencerepresented by SEQ ID NO: 19, NO: 21, NO: 23, NO: 25, NO: 27 or NO: 29.The addition of Met to the N-terminal may also enhance the antigenecityof the partial peptide. Therefore, in view of industrial applicability,it is significant to establish a method of eliminating the N-terminalMet as the initiation codon.

SUMMARY OF TE INVETION

[0007] As a result of intensive research on a method of efficientlyproducing a partial peptide of the polypeptide comprising the amino acidsequence of SEQ ID NO: 19, 21, 23, 25, 27 or 29, or a salt thereof, wediscovered that the partial peptide of the polypeptide comprising theamino acid sequence of SEQ ID NO: 19, 21, 23, 25, 27 or 29, or a saltthereof can be efficiently produced by preparing a fusion protein orpolypeptide, in which the partial peptide of the polypeptide comprisingthe amino acid sequence of SEQ ID NO: 19, 21, 23, 25, 27 or 29 isligated to the N-terminal of a protein or polypeptide having a cysteineat the N-terminal, and then subjecting the fusion protein or polypeptideto the reaction for cleavage of a peptide bond. We did further researchon the basis of the above finding and accomplished the presentinvention.

[0008] When produced according to the production method shown above, inparticular, the polypeptide having the amino acid sequence shown in SEQID NO: 3, 5, 9 or 11 may possibly have an extra methionine residue addedat the N-terminal. Thus, we made further intensive researched into amethod of producing a partial peptide of the polypeptide comprising theamino acid sequence of SEQ ID NO: 19, 21, 23, 25, 27 or 29 in a formhaving the naturally-occurring amino acid sequence, which comprisesexcising only the N-terminal methionine residue from the partial peptideof the polypeptide comprising the amino acid sequence of SEQ ID NO: 19,21, 23, 25, 27 or 29 produced by the genetic engineering. As a result,we found that, as shown in Scheme I, a transamination reaction iscarried out by reacting a partial peptide of the polypeptide comprisingthe amino acid sequence of SEQ ID NO: 19, 21, 23, 25, 27 or 29, whichhas a methionine residue optionally oxidized at the N-terminal, asrepresented by Formula I, with, for example, α-diketone such asglyoxylic acid, a donor offering transition metal ions such as coppersulfate, and a base (e.g. amines) such as pyridine; and a subsequenthydrolysis reaction is carried out by reacting the partial peptide ofthe polypeptide comprising the amino acid sequence of SEQ ID NO: 19, 21,23, 25, 27 or 29, which has the thus obtained diketone of the methionineresidue optionally oxidized at the N-terminal, with a base (e.g.diamines) such as 3,4-diamino-benzoic acid, so that the diketone ofmethionine residue can be removed unexpectedly efficiently from thepartial peptide having the diketone. This means our finding of a methodof producing unexpectedly efficiently a partial peptide of thepolypeptide comprising the amino acid sequence of SEQ ID NO: 19, 21, 23,25, 27 or 29, which does not have a methionine residue optionallyoxidized at the N-terminal, which comprises removing the N-terminalmethionine residue optionally oxidized from the partial peptide of thepolypeptide comprising the amino acid sequence of SEQ ID NO: 19, 21, 23,25, 27 or 29, which has the methionine residue, without a decrease inits activity. We finally accomplished the present invention afterfurther research thereon.

[0009] In Scheme I, m indicates an integer of 0 to 2, and X indicates apartial peptide chain of the polypeptide comprising the amino acidsequence represented by SEQ ID NO: 19, 21, 23, 25, 27 or 29.

[0010] In the present specification and Scheme I,

[0011] (1) The compound represented by the general formula (I) may referto “a partial peptide of the polypeptide comprising the amino acidsequence of SEQ ID NO: 19, 21, 23, 25, 27 or 29, which has a methionineresidue optionally oxidized at the N-tenninal, or a salt thereof” or “apartial peptide of the polypeptide comprising the amino acid sequence ofSEQ ID NO: 19, 21, 23, 25, 27 or 29, which has a methionine residue, ora salt thereof”;

[0012] (2) In the general formula (I):

[0013] wherein m is defined as above, the moiety shown above may referto “a methionine residue optionally oxidized”, “a methionine residue” or“a methionine”;

[0014] (3) The compound represented by the general formula (II) mayrefer to “a partial peptide of the polypeptide comprising the amino acidsequence of SEQ ID NO: 19, 21, 23, 25, 27 or 29, which has a diketone ofmethionine residue optionally oxidized at the N-terminal, or a saltthereof”or “the polypeptide comprising the amino acid sequence of SEQ IDNO: 19, 21, 23, 25, 27 or 29, which has a diketone of methionineresidue, or a salt thereof”;

[0015] (4) In the general formula (II) :

[0016] wherein m is defined as above, the moiety shown above may referto “a diketone of methionine residue optionally oxidized” or “a diketoneof methionine residue”;

[0017] (5) The compound represented by the general formula (III) mayrefer to “the polypeptide comprising the amino acid sequence of SEQ IDNO: 19, 21, 23, 25, 27 or 29, which does not have a methionine residueoptionally oxidized at the N-terminal, or a salt thereof” or “thepolypeptide comprising the amino acid sequence of SEQ ID NO: 19, 21, 23,25, 27 or 29, which does not have a diketone of methionine residueoptionally oxidized at the N-terminal, or a salt thereof”.

[0018] Thus, the present invention provides:

[0019] (1) A method of producing a partial peptide of the polypeptidecomprising the amino acid sequence of SEQ ID NO: 19, 21, 23, 25, 27 or29, or a salt thereof, which comprises:

[0020] subjecting a fusion protein or polypeptide, in which a partialpeptide of the polypeptide comprising the amino acid sequence of SEQ IDNO: 19, 21, 23, 25, 27 or 29, which may has a methionine residueoptionally oxidized at the N-terminal, is ligated to the N-terminal of aprotein or polypeptide having a cysteine at the N-terminal, to areaction for cleavage of a peptide bond on the amino group side of thecysteine residue.

[0021] (2) A method of producing a partial peptide of the polypeptidecomprising the amino acid sequence of SEQ ID NO: 19, 21, 23, 25, 27 or29, which may has a methionine residue at the N-terminal, or a saltthereof, which comprises:

[0022] culturing a transformant containing a vector having a DNAencoding a fusion protein or polypeptide, in which the partial peptideof the polypeptide comprising the amino acid sequence of SEQ ID NO: 19,21, 23, 25, 27 or 29, which may has a methionine residue at theN-terminal, is ligated to the N-terminal of a protein or polypeptidehaving a cysteine at the N-terminal to express the fusion protein orpolypeptide; and

[0023] subjecting the expressed fusion protein or polypeptide to areaction for cleavage of a peptide bond on the amino group side of thecysteine residue.

[0024] (3) The production method of (1) or (2), wherein the cleavagereaction is S-cyanation reaction followed by an ammonolysis orhydrolysis reaction.

[0025] (4) The production method of (1) or (2), wherein the partialpeptide of the polypeptide comprising the amino acid sequence of SEQ IDNO: 19, 21, 23, 25, 27 or 29 includes:

[0026] (i) a peptide comprising the amino acid sequence from aa 56 (Ser)to aa 92 (Phe), aa 73 (Met) to aa 92 (Phe), aa 81 (Met) to aa 92(Phe),aa 84 (Ser) to aa 92 (Phe), aa 101 (Ser) to aa 112 (Ser), aa 95 (Asn) toaa 112 (Ser), aa 115 (Asn) to aa 131 (Phe), aa 124 (Val) to aa 131(Phe), aa 1 (Met) to aa 92 (Phe), aa 1 (Met) to aa 112 (Ser), or aa 1(Met) to aa 131 (Phe) in the amino acid sequence of SEQ ID NO: 19;

[0027] (ii) a peptide comprising the amino acid sequence from aa 56(Ser) to aa 92 (Phe), aa 73 (Met) to aa 92 (Phe), aa 81 (Met) to aa 92(Phe), aa 84 (Ser) to aa 92 (Phe), aa 101 (Ser) to aa 112 (Ser), aa 95(Asn) to aa 112 (Ser), aa 115 (Asn) to aa 131 (Phe), aa 124 (Val) to aa131 (Phe), aa 1 (Met) to aa 92 (Phe), aa I (Met) to aa 112 (Ser) or aa 1(Met) to aa 131 (Phe) in the amino acid sequence of SEQ ID NO: 21;

[0028] (iii) a peptide comprising the amino acid sequence from aa 58(Ser) to aa 92 (Phe), aa 81 (Met) to aa 92 (Phe), aa 101 (Ser) to aa 112(Leu), aa 95 (Asn) to aa 112 (Leu), aa 124 (Val) to aa 131 (Phe), aa 1(Met) to aa 92 (Phe), or aa I (Met) to aa 131 (Phe) in the amino acidsequence of SEQ ID NO: 23;

[0029] (iv) a peptide comprising the amino acid sequence from aa 58(Ser) to aa 94 (Phe), aa 83 (Val) to aa 94 (Phe), aa 84 (Pro) to aa 94(Phe), or aa 118 (Phe) to aa 125 (Phe) in the amino acid sequence of SEQID NO: 25; or

[0030] (v) a peptide comprising the amino acid sequence from aa 58 (Ser)to aa 94 (Phe), or aa 84 (Pro) to aa 94 (Phe) in the amino acid sequenceof SEQ ID NO: 27.

[0031] (5) The production method of (1) or (2), wherein the partialpeptide of the polypeptide comprising the amino acid sequence of SEQ IDNO: 19, 21, 23, 25, 27 or 29 includes the polypeptide comprising theamino acid sequence of SEQ ID NO: 1 or 9.

[0032] (6) The production method of (5), wherein the polypeptidecomprising the amino acid sequence of SEQ ID NO: 1 or 9 includes thepolypeptide having the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9or 11.

[0033] (7) A fusion protein or polypeptide, in which a partial peptideof the polypeptide comprising the amino acid sequence of SEQ ID NO: 19,21, 23, 25, 27 or 29, which may has a methionine residue at theN-terminal, is ligated to the N-terminal of a protein or polypeptidehaving a cysteine at the N-terminal, or a salt thereof.

[0034] (8) The polypeptide of (7) or a salt thereof, wherein the partialpeptide of the polypeptide comprising the amino acid sequence of SEQ IDNO: 19, 21, 23, 25, 27 or 29 includes:

[0035] (i) a peptide comprising the amino acid sequence from aa 56 (Ser)to aa 92 (Phe), aa 73 (Met) to aa 92 (Phe), aa 81 (Met) to aa 92(Phe),aa 84 (Ser) to aa 92 (Phe), aa 101 (Ser) to aa 112 (Ser), aa 95 (Asn) toaa 112 (Ser), aa 115 (Asn) to aa 131 (Phe), aa 124 (Val) to aa 131(Phe), aa 1 (Met) to aa 92 (Phe), aa 1 (Met) to aa 112 (Ser), or aa 1(Met) to aa 131 (Phe) in the amino acid sequence of SEQ ID NO: 19;

[0036] (ii) a peptide comprising the amino acid sequence from aa 56(Ser) to aa 92 (Phe), aa 73 (Met) to aa 92 (Phe), aa 81 (Met) to aa 92(Phe), aa 84 (Ser) to aa 92 (Phe), aa 101 (Ser) to aa 112 (Ser), aa 95(Asn) to aa 112 (Ser), aa 115 (Asn) to aa 131 (Phe), aa 124 (Val) to aa131 (Phe), aa 1 (Met) to aa 92 (Phe), aa 1 (Met) to aa 112 (Ser) or aa 1(Met) to aa 131 (Phe) in the amino acid sequence of SEQ ID NO: 21;

[0037] (iii) a peptide comprising the amino acid sequence from aa 58(Ser) to aa 92 (Phe), aa 81 (Met) to aa 92 (Phe), aa 101 (Ser) to aa 112(Leu), aa 95 (Asn) to aa 112 (Leu), aa 124 (Val) to aa 131 (Phe), aa 1(Met) to aa 92 (Phe), or aa 1 (Met) to aa 131 (Phe) in the amino acidsequence of SEQ ID NO: 23;

[0038] (iv) a peptide comprising the amino acid sequence from aa 58(Ser) to aa 94 (Phe), aa 83 (Val) to aa 94 (Phe), aa 84 (Pro) to aa 94(Phe), or aa 118 (Phe) to aa 125 (Phe) in the amino acid sequence of SEQID NO: 25; or

[0039] (v) a peptide comprising the amino acid sequence from aa 58 (Ser)to aa 94 (Phe), or aa 84 (Pro) to aa 94 (Phe) in the amino acid sequenceof SEQ ID NO: 27.

[0040] (9) The polypeptide of (7) or a salt thereof, wherein the partialpeptide of the polypeptide comprising the amino acid sequence of SEQ IDNO: 19, 21, 23, 25, 27 or 29 includes the polypeptide comprising theamino acid sequence of SEQ ID NO: 1 or 9.

[0041] (10) The polypeptide of (9) or a salt thereof, wherein thepolypeptide comprising the amino acid sequence of SEQ ID NO: 1 or 9includes the polypeptide having the amino acid sequence of SEQ ID NO: 1,3, 5, 7, 9 or 11.

[0042] (11) A vector comprising the DNA encoding the fusion protein orpolypeptide of (7).

[0043] (12) A transformant having the vector of (11).

[0044] (13) A method of removing the N-terminal methionine residue froma partial peptide of the polypeptide comprising the amino acid sequenceof SEQ ID NO: 19, 21, 23, 25, 27 or 29, which has a methionine residueoptionally oxidized at the N-terminal, which comprises:

[0045] reacting the partial peptide or a salt thereof with α-diketone;and then

[0046] subjecting it to a hydrolysis reaction.

[0047] (14) The method of (13), wherein the partial peptide of thepolypeptide comprising the amino acid sequence of SEQ ID NO: 19, 21, 23,25, 27 or 29, which has a methionine residue optionally oxidized at theN-terminal, is produced by genetic engineering, and the amino acid nextto the methionine residue on the C-terminal side is Isoleucine, Valine,Cysteine, Threonine, Aspartic acid, Lysine, Leucine, Arginine,Asparagine, Methionine, Phenylalanine, Tyrosine, Tryptophan, Glutamicacid, Glutamine or Histidine.

[0048] (15) The method of (13) or (14), wherein the partial peptide ofthe polypeptide comprising the amino acid sequence of SEQ ID NO: 19, 21,23, 25, 27 or 29 includes:

[0049] (i) a peptide comprising the amino acid sequence from aa 56 (Ser)to aa 92 (Phe), aa 73 (Met) to aa 92 (Phe), aa 81 (Met) to aa 92(Pbe),aa 84 (Ser) to aa 92 (Phe), aa 101 (Ser) to aa 112 (Ser), aa 95 (Asn) toaa 112 (Ser), aa 115 (Asn) to aa 131 (Phe), aa 124 (Val) to aa 131(Phe), aa 1 (Met) to aa 92 (Phe), aa 1 (Met) to aa 112 (Ser), or aa 1(Met) to aa 131 (Phe) in the amino acid sequence of SEQ ID NO: 19;

[0050] (ii) a peptide comprising the amino acid sequence from aa 56(Ser) to aa 92 (Phe), aa 73 (Met) to aa 92 (Phe), aa 81 (Met) to aa 92(Phe), aa 84 (Ser) to aa 92 (Phe), aa 101 (Ser) to aa 112 (Ser), aa 95(Asn) to aa 112 (Ser), aa 115 (Asn) to aa 131 (Phe), aa 124 (Val) to aa131 (Phe), aa 1 (Met) to aa 92 (Phe), aa 1 (Met) to aa 112 (Ser) or aa 1(Met) to aa 131 (Phe) in the amino acid sequence of SEQ ID NO: 21;

[0051] (iii) a peptide comprising the amino acid sequence from aa 58(Ser) to aa 92 (Phe), aa 81 (Met) to aa 92 (Phe), aa 101 (Ser) to aa 112(Leu), aa 95 (Asn) to aa 112 (Leu), aa 124 (Val) to aa 131 (Phe), aa 1(Met) to aa 92 (Phe), or aa 1 (Met) to aa 131 (Phe) in the amino acidsequence of SEQ ID NO: 23;

[0052] (iv) a peptide comprising the amino acid sequence from aa 58(Ser) to aa 94 (Phe), aa 83 (Val) to aa 94 (Phe), aa 84 (Pro) to aa 94(Phe), or aa 118 (Phe) to aa 125 (Phe) in the amino acid sequence of SEQID NO: 25; or

[0053] (v) a peptide comprising the amino acid sequence from aa 58 (Ser)to aa 94 (Phe), or aa 84 (Pro) to aa 94 (Phe) in the amino acid sequenceof SEQ ID NO: 27.

[0054] (16) The method of (13) or (14), wherein the partial peptide ofthe polypeptide comprising the amino acid sequence of SEQ ID NO: 19, 21,23, 25, 27 or 29 includes the polypeptide comprising the amino acidsequence of SEQ ID NO: 1 or 9.

[0055] (17) The method of (16), wherein the polypeptide comprising theamino acid sequence of SEQ ID NO: 1 or 9 includes the polypeptide havingthe amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9 or 11.

[0056] (18) The method of (13), wherein the reaction with α-diketone iscarried out in the presence of a transition metal ion.

[0057] (19) The method of (13), wherein the reaction with α-diketone iscarried out in the presence of a base.

[0058] (20) The method of (13), wherein the reaction with α-diketone iscarried out in the presence of a transition metal ion and a base.

[0059] (21) The method of (13), wherein the α-diketone is glyoxylic acidor its salt.

[0060] (22) The method of (18), wherein the transition metal ion is acopper ion.

[0061] (23) The method of (19), wherein the base is pyridine.

[0062] (24) The method of (13), wherein the hydrolysis reaction iscarried out using a base.

[0063] (25) The method of (24), wherein the base is an amine.

[0064] (26) The method of (24), wherein the base is a diamine, or thio-or seleno-semicarbazide.

[0065] (27) The method of (26), wherein the diamine iso-phenylenediamine or 3,4-dianino-benzoic acid.

[0066] (28) A method of producing the polypeptide comprising the aminoacid sequence of SEQ ID NO: 1 or 9, or a salt thereof, which comprises:

[0067] reacting a partial peptide of the polypeptide comprising theamino acid sequence of SEQ ID NO: 19, 21, 23, 25, 27 or 29, which has amethionine residue added at the N-terminal, or a salt thereof, which isproduced by genetic engineering, with glyoxylic acid or its salt in thepresence of copper sulfate and pyridine; and then reacting it witho-phenylenediamine or 3,4-diamino-benzoic acid.

[0068] (29) The production method of (28), wherein the partial peptideof the polypeptide comprising the amino acid sequence of SEQ ID NO: 19,21, 23, 25, 27 or 29 includes:

[0069] (i) a peptide comprising the amino acid sequence from aa 56 (Ser)to aa 92 (Phe), aa 73 (Met) to aa 92 (Phe), aa 81 (Met) to aa 92(Phe),aa 84 (Ser) to aa 92 (Phe), aa 101 (Ser) to aa 112 (Ser), aa 95 (Asn) toaa 112 (Ser), aa 115 (Asn) to aa 131 (Phe), aa 124 (Val) to aa 131(Phe), aa 1 (Met) to aa 92 (Phe), aa 1 (Met) to aa 112 (Ser), or aa 1(Met) to aa 131 (Phe) in the amino acid sequence of SEQ ID NO: 19;

[0070] (ii) a peptide comprising the amino acid sequence from aa 56(Ser) to aa 92 (Phe), aa 73 (Met) to aa 92 (Phe), aa 81 (Met) to aa 92(Phe), aa 84 (Ser) to aa 92 (Phe), aa 101 (Ser) to aa 112 (Ser), aa 95(Asn) to aa 112 (Ser), aa 115 (Asn) to aa 131 (Phe), aa 124 (Val) to aa131 (Phe), aa 1 (Met) to aa 92 (Phe), aa 1 (Met) to aa 112 (Ser) or aa 1(Met) to aa 131 (Phe) in the amino acid sequence of SEQ ID NO: 21;

[0071] (iii) a peptide comprising the amino acid sequence from aa 58(Ser) to aa 92 (Phe), aa 81 (Met) to aa 92 (Phe), aa 101 (Ser) to aa 112(Leu), aa 95 (Asn) to aa 112 (Leu), aa 124 (Val) to aa 131 (Phe), aa 1(Met) to aa 92 (Phe), or aa 1 (Met) to aa 131 (Phe) in the amino acidsequence of SEQ ID NO: 23;

[0072] (iv) a peptide comprising the amino acid sequence from aa 58(Ser) to aa 94 (Phe), aa 83 (Val) to aa 94 (Phe), aa 84 (Pro) to aa 94(Phe), or aa 118 (Phe) to aa 125 (Phe) in the amino acid sequence of SEQID NO: 25; or

[0073] (v) a peptide comprising the amino acid sequence from aa 58 (Ser)to aa 94 (Phe), or aa 84 (Pro) to aa 94 (Phe) in the amino acid sequenceof SEQ ID NO: 27.

[0074] (30) The production method of (28), wherein the partial peptideof the polypeptide comprising the amino acid sequence of SEQ ID NO: 19,21, 23, 25, 27 or 29 includes the polypeptide comprising the amino acidsequence of SEQ ID NO: 1 or 9.

[0075] (31) The production method of (30), wherein the polypeptidecomprising the amino acid sequence of SEQ ID NO: 1 or 9 includes thepolypeptide having the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9or 11.

[0076] (32) A compound represented by the formulaCH₃—S(O)_(m)—(CH₂)₂—CO—CO—X, or a salt thereof, wherein m indicates aninteger of 0 to 2, and X indicates a partial peptide chain of thepolypeptide comprising the amino acid sequence of SEQ ID NO: 19, 21, 23,25, 27 or 29.

[0077] (33) The compound of (32), or a salt thereof, wherein the partialpeptide of the polypeptide comprising the amino acid sequence of SEQ IDNO: 19, 21, 23, 25, 27 or 29 includes:

[0078] (i) a peptide comprising the amino acid sequence from aa 56 (Ser)to aa 92 (Phe), aa 73 (Met) to aa 92 (Phe), aa 81 (Met) to aa 92(Phe),aa 84 (Ser) to aa 92 (Phe), aa 101 (Ser) to aa 112 (Ser), aa 95 (Asn) toaa 112 (Ser), aa 115 (Asn) to aa 131 (Phe), aa 124 (Val) to aa 131(Phe), aa 1 (Met) to aa 92 (Phe), aa 1 (Met) to aa 112 (Ser), or aa 1(Met) to aa 131 (Phe) in the amino acid sequence of SEQ ID NO: 19;

[0079] (ii) a peptide comprising the amino acid sequence from aa 56(Ser) to aa 92 (Phe), aa 73 (Met) to aa 92 (Phe), aa 81 (Met) to aa 92(Phe), aa 84 (Ser) to aa 92 (Phe), aa 101 (Ser) to aa 112 (Ser), aa 95(Asn) to aa 112 (Ser), aa 115 (Asn) to aa 131 (Phe), aa 124 (Val) to aa131 (Phe), aa 1 (Met) to aa 92 (Phe), aa 1 (Met) to aa 112 (Ser) or aa 1(Met) to aa 131 (Phe) in the amino acid sequence of SEQ ID NO: 21;

[0080] (iii) a peptide comprising the amino acid sequence from aa 58(Ser) to aa 92 (Phe), aa 81 (Met) to aa 92 (Phe), aa 101 (Ser) to aa 112(Leu), aa 95 (Asn) to aa 112 (Leu), aa 124 (Val) to aa 131 (Phe), aa 1(Met) to aa 92 (Phe), or aa 1 (Met) to aa 131 (Phe) in the amino acidsequence of SEQ ID NO: 23;

[0081] (iv) a peptide comprising the amino acid sequence from aa 58(Ser) to aa 94 (Phe), aa 83 (Val) to aa 94 (Phe), aa 84 (Pro) to aa 94(Phe), or aa 118 (Phe) to aa 125 (Phe) in the amino acid sequence of SEQID NO: 25; or

[0082] (v) a peptide comprising the amino acid sequence from aa 58 (Ser)to aa 94 (Phe), or aa 84 (Pro) to aa 94 (Phe) in the amino acid sequenceof SEQ ID NO: 27.

[0083] (34) The compound of (32), or a salt thereof, wherein the partialpeptide of the polypeptide comprising the amino acid sequence of SEQ IDNO: 19, 21, 23, 25, 27 or 29 includes the polypeptide comprising theamino acid sequence of SEQ ID NO: 1 or 9.

[0084] (35) The compound of (34), or a salt thereof, wherein thepolypeptide comprising the amino acid sequence of SEQ ID NO: 1 or 9includes the polypeptide having the amino acid sequence of SEQ ID NO: 1,3, 5, 7, 9 or 11.

[0085] (36) A method of producing a partial peptide of the polypeptidecomprising the amino acid sequence of SEQ ID NO: 19, 21, 23, 25, 27 or29, or a salt thereof, which comprises subjecting the compound of (32)to a hydrolysis reaction.

[0086] (37) A method of producing the polypeptide comprising the aminoacid sequence represented by, or a salt thereof, which comprises:

[0087] subjecting a fusion protein or polypeptide, in which thepolypeptide comprising the amino acid sequence represented by, which hasa methionine residue at the N-terminal, is ligated to the N-terminal ofa protein or polypeptide having a cysteine at the N-terminal, to areaction for cleavage of a peptide bond on the amino group side of thecysteine residue to obtain the polypeptide comprising the amino acidsequence represented by, which has a methionine residue optionallyoxidized at the N-terminal, or a salt thereof;

[0088] reacting the polypeptide comprising the amino acid sequencerepresented by, which has a methionine residue optionally oxidized atthe N-terminal, or a salt thereof with α-diketone; and

[0089] subjecting it to a hydrolysis reaction.

[0090] (38) The production method of (37), wherein the partial peptideof the polypeptide comprising the amino acid sequence of SEQ ID NO: 19,21, 23, 25, 27 or 29 includes:

[0091] (i) a peptide comprising the amino acid sequence from aa 56 (Ser)to aa 92 (Phe), aa 73 (Met) to aa 92 (Phe), aa 81 (Met) to aa 92(Phe),aa 84 (Ser) to aa 92 (Phe), aa 101 (Ser) to aa 112 (Ser), aa 95 (Asn) toaa 112 (Ser), aa 115 (Asn) to aa 131 (Phe), aa 124 (Val) to aa 131(Phe), aa 1 (Met) to aa 92 (Phe), aa 1 (Met) to aa 112 (Ser), or aa 1(Met) to aa 131 (Phe) in the amino acid sequence of SEQ ID NO: 19;

[0092] (ii) a peptide comprising the amino acid sequence from aa 56(Ser) to aa 92 (Phe), aa 73 (Met) to aa 92 (Phe), aa 81 (Met) to aa 92(Phe), aa 84 (Ser) to aa 92 (Phe), aa 101 (Ser) to aa 112 (Ser), aa 95(Asn) to aa 112 (Ser), aa 115 (Asn) to aa 131 (Phe), aa 124 (Val) to aa131 (Phe), aa 1 (Met) to aa 92 (Phe), aa 1 (Met) to aa 112 (Ser) or aa 1(Met) to aa 131 (Phe) in the amino acid sequence of SEQ ID NO: 21;

[0093] (iii) a peptide comprising the amino acid sequence from aa 58(Ser) to aa 92 (Phe), aa 81 (Met) to aa 92 (Phe), aa 101 (Ser) to aa 112(Leu), aa 95 (Asn) to aa 112 (Leu), aa 124 (Val) to aa 131 (Phe), aa 1(Met) to aa 92 (Phe), or aa 1 (Met) to aa 131 (Phe) in the amino acidsequence of SEQ ID NO: 23;

[0094] (iv) a peptide comprising the amino acid sequence from aa 58(Ser) to aa 94 (Phe), aa 83 (Val) to aa 94 (Phe), aa 84 (Pro) to aa 94(Phe), or aa 118 (Phe) to aa 125 (Phe) in the amino acid sequence of SEQID NO: 25; or

[0095] (v) a peptide comprising the amino acid sequence from aa 58 (Ser)to aa 94 (Phe), or aa 84 (Pro) to aa 94 (Phe) in the amino acid sequenceof SEQ ID NO: 27.

[0096] (39) The production method of (37), wherein the partial peptideof the polypeptide comprising the amino acid sequence of SEQ ID NO: 19,21, 23, 25, 27 or 29 includes the polypeptide comprising the amino acidsequence of SEQ ID NO: 1 or 9.

[0097] (40) The production method of (39), wherein the polypeptidecomprising the amino acid sequence of SEQ ID NO: 1 or 9 includes thepolypeptide having the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9or 11.

BRIEF DESCRIPTION OF THE DRAWINGS

[0098]FIG. 1 indicates the reaction mechanism in the production methodof the invention.

[0099]FIG. 2 indicates a schematic diagram of the construction of theplasmid pTFCRFRP-1 obtained in Example 2.

BEST MODE FOR CARRYING OUT THE INVENTION

[0100] The polypeptides comprising the amino acid sequence representedby SEQ ID NO: 19, NO: 21, NO: 23, NO: 25, NO: 27 or NO: 29 in theinvention (sometimes referred to as the precursor polypeptide of theinvention) are polypeptides comprising the amino acid sequencerepresented by SEQ ID NO: 1, NO: 8, NO: 14, NO: 33, NO: 50 or NO: 19,respectively, described in WO 00/29441 and WO 01/66134.

[0101] The precursor polypeptides of the invention are shown accordingto the conventional notation so that the N-terminal (amino terminal) isplaced on the left side and the C-tenrinal (carboxyl terminal) on theright side. In the precursor polypeptides of the invention, includingthe precursor polypeptide comprising the amino acid sequence shown bySEQ ID NO: 1, their C-terminals are normally carboxyl group (—COOH) orcarboxylate (—COO⁻), and may also be amide (—CONH₂) or ester (—COOR).

[0102] The ester group “R” includes C₁₋₆ alkyl group such as methyl,ethyl, n-propyl, isopropyl, or n-butyl; C₃₋₈ cycloalkyl group such ascyclopenthyl, or cyclohexyl; C₆₋₁₂ aryl group such as phenyl, orα-naphthyl; C₇₋₁₄ aralkyl group such as phenyl-C₁₋₂ alkyl, e.g. benzylor phenethyl, or α-naphthyl-C₁₋₂ alkyl, e.g. α-naphthylmethyl; orpivaloyloxymethyl group generally used as an oral ester.

[0103] When the precursor polypeptide of the invention has a carboxylgroup or carboxylate at a position other than the C-terminal, it may beamidated or esterified. Such an amide or ester foirn is also included inthe polypeptide of the invention. The ester group may be the same asdescribed with respect to the above C-terminal ester group.

[0104] Furthermore, the precursor polypeptide of the invention includesvariants thereof, wherein the amino group at the N-terminal (e.g.,methionine residue) is protected with a protecting group (e.g., C₁₋₆acyl group such as C₁₋₆ alkanoyl, e.g. formyl group, acetyl group,etc.); those wherein the N-terminal region is cleaved in vivo and theglutamyl group thus formed is pyroglutaminated; those wherein asubstituent (e.g., —OH, —SH, amino group, imidazole group, indole group,guanidino group, etc.) on the side chain of an amino acid in themolecule is protected with a suitable protecting group (e.g., C₁₋₆ acylgroup such as C₁₋₆ alkanoyl group, e.g., formyl group, acetyl group,etc.), or conjugated proteins such as a glycoprotein having sugarchains. Hereinafter, the term “the precursor polypeptide of theinvention” may be used to include these polypeptides.

[0105] The partial peptide of the precursor polypeptide of the invention(hereinafter occasionally referred to as the polypeptides of theinvention) may be any partial peptides of the precursor polypeptide ofthe invention described above, for example, any one which can bind to areceptor protein of the precursor polypeptide (specifically, the proteincomprising the same or substantially the same amino acid sequence asthat shown in SEQ ID NO: 37 in WO 00/29441 and WO 01/66134, or a saltthereof) and has an activity of regulating prolactin secretion.

[0106] In the polypeptide of the invention, 1 to 5 (preferably 1 to 3)amino acids in the amino acid sequence of the polypeptide may bedeleted; or 1 to 5 (preferably 1 to 3) amino acids may be added to theamino acid sequence; or 1 to 5 (preferably 1 to 3) amino acids may beinserted into the amino acid sequence; or 1 to 5 (preferably 1 to 3)amino acids in the amino acid sequence may be substituted by other aminoacids; or a combination thereof may be included in the amino acidsequence.

[0107] In the polypeptides of the invention, the C-terminals arenormally carboxyl group (—COOH) or carboxylate (—COO⁻), and may also beamide (—CONH₂) or ester (—COOR), as described in the precursorpolypeptide of the invention (R is defined as described above).Particularly preferred is the polypeptide having amidated C-terminal(—CONH₂).

[0108] When the polypeptide of the invention has a carboxyl group (orcarboxylate) at a position other than the C-terminal, it may be amidatedor esterified. Such an amide or ester form is also included in thepolypeptide of the invention. The ester group may be the same asdescribed with respect to the above C-terminal ester group.

[0109] Furthermore, as well as the precursor polypeptide of theinvention as described above, the polypeptide of the invention includesvariants thereof, wherein the amino group at the N-terminal (e.g.,methionine residue) is protected with a protecting group; those whereinthe N-terminal region is cleaved in vivo and the glutamyl group thusformed is pyroglutaminated; those wherein a substituent on the sidechain of an amino acid in the molecule is protected with a suitableprotecting group, or conjugated proteins such as a glycoprotein havingsugar chains. Hereinafter, the term “the polypeptide of the invention”may be used to include these polypeptides.

[0110] The partial peptide of the precursor polypeptide of the inventionmay be, for example, a peptide having RFamide, RS amide or RLamidestructure, more preferably, a peptide having RFamide or RS amidestructure, and particularly preferably, a peptide having RFamidestructure.

[0111] The RFamide structure means that the C-terminal of the peptide isArg-Phe-NH₂. The RSamide structure means that the C-terminal of thepeptide is Arg-Ser-NH₂. The RLamide structure means that the C-terminalof the peptide is Arg-Leu-NH₂.

[0112] Preferred examples of the polypeptide of the invention include:

[0113] (1) a peptide comprising the amino acid sequence from aa 56 (Ser)to aa 92 (Phe), aa 73 (Met) to aa 92 (Phe), aa 81 (Met) to aa 92(Phe),aa 84 (Ser) to aa 92 (Phe), aa 101 (Ser) to aa 112 (Ser), aa 95 (Asn) toaa 112 (Ser), aa 115 (Asn) to aa 131 (Phe), aa 124 (Val) to aa 131(Phe), aa 1 (Met) to aa 92 (Phe), aa 1 (Met) to aa 112 (Ser), or aa 1(Met) to aa 131 (Phe) in the amino acid sequence of SEQ ID NO: 19;

[0114] (2) a peptide comprising the amino acid sequence from aa 56 (Ser)to aa 92 (Phe), aa 73 (Met) to aa 92 (Phe), aa 81 (Met) to aa 92 (Phe),aa 84 (Ser) to aa 92 (Phe), aa 101 (Ser) to aa 112 (Ser), aa 95 (Asn) toaa 112 (Ser), aa 115 (Asn) to aa 131 (Phe), aa 124 (Val) to aa 131(Phe), aa 1 (Met) to aa 92 (Phe), aa 1 (Met) to aa 112 (Ser) or aa 1(Met) to aa 131 (Phe) in the amino acid sequence of SEQ ID NO: 21;

[0115] (3) a peptide comprising the amino acid sequence from aa 58 (Ser)to aa 92 (Phe), aa 81 (Met) to aa 92 (Phe), aa 101 (Ser) to aa 112(Leu), aa 95 (Asn) to aa 112 (Leu), aa 124 (Val) to aa 131 (Phe), aa 1(Met) to aa 92 (Phe), or aa 1 (Met) to aa 131 (Phe) in the amino acidsequence of SEQ ID NO: 23;

[0116] (4) a peptide comprising the amino acid sequence from aa 58 (Ser)to aa 94 (Phe), aa 83 (Val) to aa 94 (Phe), aa 84 (Pro) to aa 94 (Phe),or aa 118 (Phe) to aa 125 (Phe) in the amino acid sequence of SEQ ID NO:25;

[0117] (5) a peptide comprising the amino acid sequence from aa 58 (Ser)to aa 94 (Phe) or aa 84 (Pro) to aa 94 (Phe) in the amino acid sequenceof SEQ ID NO: 27.

[0118] In particular, preferred examples include:

[0119] (1) a peptide comprising the amino acid sequence from aa 56 (Ser)to aa 92 (Phe), aa 73 (Met) to aa 92 (Phe), aa 81 (Met) to aa 92(Phe),aa 84 (Ser) to aa 92 (Phe), aa 101 (Ser) to aa 112 (Ser), aa 95 (Asn) toaa 112 (Ser), aa 115 (Asn) to aa 131 (Phe), or aa 124 (Val) to aa 131(Phe) in the amino acid sequence of SEQ ID NO: 19;

[0120] (2) a peptide comprising the amino acid sequence from aa 56 (Ser)to aa 92 (Phe), aa 73 (Met) to aa 92 (Phe), aa 81 (Met) to aa 92 (Phe),aa 84 (Ser) to aa 92 (Phe), aa 101 (Ser) to aa 112 (Ser), aa 95 (Asn) toaa 112 (Ser), aa 115 (Asn) to aa 131 (Phe), or aa 124 (Val) to aa 131(Phe) in the amino acid sequence of SEQ ID NO: 21;

[0121] (3) a peptide comprising the amino acid sequence from aa 58 (Ser)to aa 92 (Phe), aa 81 (Met) to aa 92 (Phe), aa 101 (Ser) to aa 112(Leu), aa 95 (Asn) to aa 112 (Leu), or aa 124 (Val) to aa 131 (Phe) inthe amino acid sequence of SEQ ID NO: 23;

[0122] (4) a peptide comprising the amino acid sequence from aa 58 (Ser)to aa 94 (Phe), aa 83 (Val) to aa 94 (Phe) or aa 118 (Phe) to aa 125(Phe) in the amino acid sequence of SEQ ID NO: 25;

[0123] (5) a peptide comprising the amino acid sequence from aa 58 (Ser)to aa 94 (Phe) in the amino acid sequence of SEQ ID NO: 27.

[0124] In particular, preferred examples include:

[0125] (1) a peptide comprising the amino acid sequence from aa 56 (Ser)to aa 92 (Phe), aa 73 (Met) to aa 92 (Phe), aa 81 (Met) to aa 92(Phe),aa 84 (Ser) to aa 92 (Phe), aa 115 (Asn) to aa 131 (Phe), or aa 124(Val) to aa 131 (Phe) in the amino acid sequence of SEQ ID NO: 19;

[0126] (2) a peptide comprising the amino acid sequence from aa 56 (Ser)to aa 92 (Phe), aa 73 (Met) to aa 92 (Phe), aa 81 (Met) to aa 92 (Phe),aa 84 (Ser) to aa 92 (Phe), aa 115 (Asn) to aa 131 (Phe), or aa 124(Val) to aa 131 (Phe) in the amino acid sequence of SEQ ID NO: 21;

[0127] (3) a peptide comprising the amino acid sequence from aa 58 (Ser)to aa 92 (Phe), aa 81 (Met) to aa 92 (Phe) or aa 124 (Val) to aa 131(Phe) in the amino acid sequence of SEQ ID NO: 23.

[0128] In particular, preferred examples include:

[0129] (1) a peptide comprising the amino acid sequence from aa 56 (Ser)to aa 92 (Phe), aa 73 (Met) to aa 92 (Phe), aa 81 (Met) to aa 92(Phe),or aa 84 (Ser) to aa 92 (Phe) in the amino acid sequence of SEQ ID NO:19;

[0130] (2) a peptide comprising the amino acid sequence from aa 56 (Ser)to aa 92 (Phe), aa 73 (Met) to aa 92 (Phe), aa 81 (Met) to aa 92 (Phe),or aa 84 (Ser) to aa 92 (Phe) in the amino acid sequence of SEQ ID NO:21;

[0131] (3) a peptide comprising the amino acid sequence from aa 58 (Ser)to aa 92 (Phe) or aa 81 (Met) to aa 92 (Phe) in the amino acid sequenceof SEQ ID NO: 23.

[0132] In particular, preferred examples include:

[0133] (1) a peptide comprising the amino acid sequence from aa 81 (Met)to aa 92(Phe) in the amino acid sequence of SEQ ID NO: 19;

[0134] (2) a peptide comprising the amino acid sequence from aa 81 (Met)to aa 92 (Phe) in the amino acid sequence of SEQ ID NO: 21;

[0135] (3) a peptide comprising the amino acid sequence from aa 81 (Met)to aa 92 (Phe) in the amino acid sequence of SEQ ID NO: 23.

[0136] In particular, preferred examples include:

[0137] (1) a peptide comprising the amino acid sequence from aa 81 (Met)to aa 92(Phe) in the amino acid sequence of SEQ ID NO: 19;

[0138] (2) a peptide comprising the amino acid sequence from aa 81 (Met)to aa 92 (Phe) in the amino acid sequence of SEQ ID NO: 21.

[0139] Further, preferred examples of the polypeptide of the inventioninclude:

[0140] (a) a peptide having the amino acid sequence from aa 81 (Met) toaa 92 (Phe) in the amino acid sequence of SEQ ID NO: 19;

[0141] (b) a peptide having the amino acid sequence from aa 101 (Ser) toaa 112 (Ser) in the amino acid sequence of SEQ ID NO: 19;

[0142] (c) a peptide having the amino acid sequence from aa 124 (Val) toaa 131 (Phe) in the amino acid sequence of SEQ ID NO: 19;

[0143] (d) a peptide having the amino acid sequence from aa 56 (Ser) toaa 92 (Phe) in the amino acid sequence of SEQ ID NO: 19;

[0144] (e) a peptide having the amino acid sequence from aa 81 (Met) toaa 92 (Phe) in the amino acid sequence of SEQ ID NO: 23;

[0145] (f) a peptide having the amino acid sequence from aa 101 (Ser) toaa 112 (Leu) in the amino acid sequence of SEQ ID NO: 23;

[0146] (g) a peptide having the amino acid sequence from aa 58 (Ser) toaa 92 (Phe) in the amino acid sequence of SEQ ID NO: 23;

[0147] (h) a peptide having the amino acid sequence from aa 83 (Val) toaa 94 (Phe) in the amino acid sequence of SEQ ID NO: 25;

[0148] (i) a peptide having the amino acid sequence from aa 118 (Phe) toaa 125 (Phe) in the amino acid sequence of SEQ ID NO: 25;

[0149] (j) a peptide having the amino acid sequence from aa 58 (Ser) toaa 94 (Phe) in the amino acid sequence of SEQ ID NO: 25;

[0150] (k) a peptide having the amino acid sequence from aa 58 (Ser) toaa 94 (Phe) in the amino acid sequence of SEQ ID NO: 27.

[0151] In particular, these peptides are preferably in the amide form(wherein, preferably, the C-terminal carboxyl group (—COOH) is amidated(—CONH₂)).

[0152] Examples of these peptides in the amide form include:

[0153] a peptide having the amino acid sequence from aa 81 (Met) to aa92 (Phe) in the amino acid sequence of SEQ ID NO: 19, wherein theC-terminal is amidated (—CONH₂);

[0154] a peptide having the amino acid sequence from aa 101 (Ser) to aa112 (Ser) in the amino acid sequence of SEQ ID NO: 19, wherein theC-terminal is amidated (—CONH₂);

[0155] a peptide having the amino acid sequence from aa 124 (Val) to aa131 (Phe) in the amino acid sequence of SEQ ID NO: 19, wherein theC-terminal is amidated (—CONH₂).

[0156] Among them, preferred are a peptide having the amino acidsequence from aa 81 (Met) to aa 92 (Phe) in the amino acid sequence ofSEQ ID NO: 19, wherein the C-terminal is amidated (—CONH₂); and apeptide having the amino acid sequence from aa 124 (Val) to aa 131 (Phe)in the amino acid sequence of SEQ ID NO: 19, wherein the C-terminal isamidated (—CONH₂). In particular, preferred is a peptide having theamino acid sequence from aa 81 (Met) to aa 92 (Phe) in the amino acidsequence of SEQ ID NO: 19, wherein the C-terminal is amidated (—CONH₂).

[0157] In addition, as the polypeptide of the invention, for example, apolypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 or9 may preferably be used. Specifically, any polypeptides comprising theamino acid sequence shown in SEQ ID NO: 1 or 9, for example,RFamide-related peptides described in Hinuma et al., Nature CellBiology, Vol.2, p.703-708 (2000) and polypeptides described in WO00/29441, and having an ligand activity to the receptor 0T7T022described in Hinuma et al., Nature Cell Biology, Vol.2, p.703-708 (2000)and WO 00/29441 may be used.

[0158] Such polypeptides include the polypeptide having the amino acidsequence represented by SEQ ID NO: 1, 3, 5, 7, 9 or 11.

[0159] Examples of the DNA encoding the precursor polypeptide of theinvention include:

[0160] (1) For the DNA encoding the human precursor polypeptidecomprising the amino acid sequence of SEQ ID NO: 19, a DNA comprisingthe nucleotide sequence of SEQ ID NO: 20;

[0161] (2) For the DNA encoding the human precursor polypeptidecomprising the amino acid sequence of SEQ ID NO: 21, a DNA comprisingthe nucleotide sequence of SEQ ID NO: 22;

[0162] (3) For the DNA encoding the bovine precursor polypeptidecomprising the amino acid sequence of SEQ ID NO: 23, a DNA comprisingthe nucleotide sequence of SEQ ID NO: 24;

[0163] (4) For the DNA encoding the mouse precursor polypeptidecomprising the amino acid sequence of SEQ ID NO: 25, a DNA comprisingthe nucleotide sequence of SEQ ID NO: 26;

[0164] (5) For the DNA encoding the rat precursor polypeptide comprisingthe amino acid sequence of SEQ ID NO: 27, a DNA comprising thenucleotide sequence of SEQ ID NO: 28;

[0165] (6) For the DNA encoding the rat precursor polypeptide comprisingthe amino acid sequence of SEQ ID NO: 29, a DNA comprising thenucleotide sequence of SEQ ID NO: 30.

[0166] The DNA encoding the polypeptide of the invention may be a DNAhaving a partial nucleotide sequence of the DNA comprising the nuleoitdesequence represented by SEQ ID NO: 20, 22, 24, 26, 28 or 30.

[0167] Examples of the DNA encoding the partial peptide of the inventioninclude:

[0168] (1) For the DNA encoding a peptide comprising the sequence fromaa 81 (Met) to aa 92(Phe) in the amino acid sequence of SEQ ID NO: 19,the DNA comprising the sequence from base 241 to base 276 in the basesequence of SEQ ID NO: 20;

[0169] (2) For the DNA encoding a peptide comprising the sequence fromaa 101 (Ser) to aa 112 (Ser) in the amino acid sequence of SEQ ID NO:19, the DNA comprising the sequence from base 301 to base 336 in thebase sequence of SEQ ID NO: 20;

[0170] (3) For the DNA encoding a peptide comprising the sequence fromaa 124 (Val) to aa 131 (Phe) in the amino acid sequence of SEQ ID NO:19, the DNA comprising the sequence from base 370 to base 393 in thebase sequence of SEQ ID NO: 20;

[0171] (4) For the DNA encoding a peptide comprising the sequence fromaa 1 (Met) to aa 92 (Phe) in the amino acid sequence of SEQ ID NO: 19,the DNA comprising the sequence from base 1 to base 276 in the basesequence of SEQ ID NO: 20;

[0172] (5) For the DNA encoding a peptide comprising the sequence fromaa 1 (Met) to aa 112 (Ser) in the amino acid sequence of SEQ ID NO: 19,the DNA comprising the sequence from base 1 to base 336 in the basesequence of SEQ ID NO: 20;

[0173] (6) For the DNA encoding a peptide comprising the sequence fromaa 1 (Met) to aa 131 (Phe) in the amino acid sequence of SEQ ID NO: 19,the DNA comprising the sequence from base 1 to base 393 in the basesequence of SEQ ID NO: 20;

[0174] (7) For the DNA encoding a peptide comprising the sequence fromaa 56 (Ser) to aa 92 (Phe) in the amino acid sequence of SEQ ID NO: 19,the DNA comprising the sequence from base 166 to base 276 in the basesequence of SEQ ID NO: 20;

[0175] (8) For the DNA encoding a peptide comprising the sequence fromaa 73 (Met) to aa 92 (Phe) in the amino acid sequence of SEQ ID NO: 19,the DNA comprising the sequence from base 217 to base 276 in the basesequence of SEQ ID NO: 20;

[0176] (9) For the DNA encoding a peptide comprising the sequence fromaa 84 (Ser) to aa 92 (Phe) in the amino acid sequence of SEQ ID NO: 19,the DNA comprising the sequence from base 253 to base 276 in the basesequence of SEQ ID NO: 20;

[0177] (10) For the DNA encoding a peptide comprising the sequence fromaa 95 (Asn) to aa 112 (Ser) in the amino acid sequence of SEQ ID NO: 19,the DNA comprising the sequence from base 283 to base 393 in the basesequence of SEQ ID NO: 20;

[0178] (11) For the DNA encoding a peptide comprising the sequence fromaa 115 (Asn) to aa 131 (Phe) in the amino acid sequence of SEQ ID NO:19, the DNA comprising the sequence from base 346 to base 393 in thebase sequence of SEQ ID NO: 20;

[0179] (12) For the DNA encoding a peptide comprising the sequence fromaa 56 (Ser) to aa 92 (Phe) in the amino acid sequence of SEQ ID NO: 21,the DNA comprising the sequence from base 169 to base 276 in the basesequence of SEQ ID NO: 22;

[0180] (13) For the DNA encoding a peptide comprising the sequence fromaa 73 (Met) to aa 92 (Phe) in the amino acid sequence of SEQ ID NO: 21,the DNA comprising the sequence from base 220 to base 276 in the basesequence of SEQ ID NO: 22;

[0181] (14) For the DNA encoding a peptide comprising the sequence fromaa 81 (Met) to aa 92 (Phe) in the amino acid sequence of SEQ ID NO: 21,the DNA comprising the sequence from base 244 to base 276 in the basesequence of SEQ ID NO: 22;

[0182] (15) For the DNA encoding a peptide comprising the sequence fromaa 84 (Ser) to aa 92 (Phe) in the amino acid sequence of SEQ ID NO: 21,the DNA comprising the sequence from base 253 to base 276 in the basesequence of SEQ ID NO: 22;

[0183] (16) For the DNA encoding a peptide comprising the sequence fromaa 101 (Ser) to aa 112 (Ser) in the amino acid sequence of SEQ ID NO:21, the DNA comprising the sequence from base 304 to base 336 in thebase sequence of SEQ ID NO: 22;

[0184] (17) For the DNA encoding a peptide comprising the sequence fromaa 95 (Asn) to aa 112 (Ser) in the amino acid sequence of SEQ ID NO: 21,the DNA comprising the sequence from base 283 to base 393 in the basesequence of SEQ ID NO: 22;

[0185] (18) For the DNA encoding a peptide comprising the sequence fromaa 115 (Asn) to aa 131 (Phe) in the amino acid sequence of SEQ ID NO:21, the DNA comprising the sequence from base 346 to base 393 in thebase sequence of SEQ ID NO: 22;

[0186] (19) For the DNA encoding a peptide comprising the sequence fromaa 124 (Val) to aa 131 (Phe) in the amino acid sequence of SEQ ID NO:21, the DNA comprising the sequence from base 373 to base 393 in thebase sequence of SEQ ID NO: 22;

[0187] (20) For the DNA encoding a peptide comprising the sequence fromaa 1 (Met) to aa 92 (Phe) in the amino acid sequence of SEQ ID NO: 21,the DNA comprising the sequence from base 1 to base 276 in the basesequence of SEQ ID NO: 22;

[0188] (21) For the DNA encoding a peptide comprising the sequence fromaa 1 (Met) to aa 112 (Ser) in the amino acid sequence of SEQ ID NO: 21,the DNA comprising the sequence from base 1 to base 336 in the basesequence of SEQ ID NO: 22;

[0189] (22) For the DNA encoding a peptide comprising the sequence fromaa 1 (Met) to aa 131 (Phe) in the amino acid sequence of SEQ ID NO: 21,the DNA comprising the sequence from base 1 to base 393 in the basesequence of SEQ ID NO: 22;

[0190] (23) For tile DNA encoding a peptide comprising the sequence fromaa 58 (Ser) to aa 92 (Phle) in the amino acid sequence of SEQ ID NO: 23,the DNA comprising the sequence from base 172 to base 276 in tile basesequence of SEQ ID NO: 24;

[0191] (24) For the DNA encoding a peptide comprising the sequence fromaa 81 (Met) to aa 92 (Phe) in the amino acid sequence of SEQ ID NO: 23,the DNA comprising the sequence from base 241 to base 276 in the basesequence of SEQ ID NO: 24;

[0192] (25) For the DNA encoding a peptide comprising the sequence fromaa 101 (Ser) to aa 112 (Leu) in the amino acid sequence of SEQ ID NO:23, the DNA comprising the sequence from base 301 to base 336 in thebase sequence of SEQ ID NO: 24;

[0193] (26) For the DNA encoding a peptide comprising the sequence fromaa 95 (Asn) to aa 112 (Leu) in the amino acid sequence of SEQ ID NO: 23,the DNA comprising the sequence from base 283 to base 336 in the basesequence of SEQ ID NO: 24;

[0194] (27) For the DNA encoding a peptide comprising the sequence fromaa 124 (Val) to aa 131 (Phe) in the amino acid sequence of SEQ ID NO:23, the DNA comprising the sequence from base 370 to base 393 in thebase sequence of SEQ ID NO: 24;

[0195] (28) For the DNA encoding a peptide comprising the sequence fromaa 1 (Met) to aa 92 (Phe) in the amino acid sequence of SEQ ID NO: 23,the DNA comprising the sequence from base 1 to base 276 in the basesequence of SEQ ID NO: 24;

[0196] (29) For the DNA encoding a peptide comprising the sequence fromaa 1 (Met) to aa 131 (Phe) in the amino acid sequence of SEQ ID NO: 23,the DNA comprising the sequence from base 1 to base 393 in the basesequence of SEQ ID NO: 24;

[0197] (30) For the DNA encoding a peptide comprising the sequence fromaa 58 (Ser) to aa 94 (Phe) in the amino acid sequence of SEQ ID NO: 25,the DNA comprising the sequence from base 172 to base 282 in the basesequence of SEQ ID NO: 26;

[0198] (31) For the DNA encoding a peptide comprising the sequence fromaa 83 (Val) to aa 94 (Phe) in the amino acid sequence of SEQ ID NO: 25,the DNA comprising the sequence from base 247 to base 282 in the basesequence of SEQ ID NO: 26;

[0199] (32) For the DNA encoding a peptide comprising the sequence fromaa 84 (Pro) to aa 94 (Phe) in the amino acid sequence of SEQ ID NO: 25,the DNA comprising the sequence from base 250 to base 282 in the basesequence of SEQ ID NO: 26;

[0200] (33) For the DNA encoding a peptide comprising the sequence fromaa 118 (Phe) to aa 125 (Phe) in the amino acid sequence of SEQ ID NO:25, the DNA comprising the sequence from base 352 to base 375 in thebase sequence of SEQ ID NO: 26;

[0201] (34) For the DNA encoding a peptide comprising the sequence fromaa 58 (Ser) to aa 94 (Phe) in the amino acid sequence of SEQ ID NO: 27,the DNA comprising the sequence from base 172 to base 282 in the basesequence of SEQ ID NO: 28;

[0202] (35) For the DNA encoding a peptide comprising the sequence fromaa 84 (Pro) to aa 94 (Phe) in the amino acid sequence of SEQ ID NO: 27,the DNA comprising the sequence from base 250 to base 282 in the basesequence of SEQ ID NO: 28.

[0203] The protein or polypeptide having cysteine at its N-terminus canbe used in the method of the invention without a particular limitation.A protein or polypeptide not having cysteine at its N-terminus may alsobe used after modification so that it will have cysteine at itsN-terminus by an ordinary method.

[0204] The protein or polypeptide having a cysteine at the N-terminalpreferably has a molecular weight of 100 to 100,000, and more preferablya molecular weight of 300 to 50,000. In addition, the protein orpolypeptide having a cysteine at the N-terminal preferably has 1 to1,000 amino acids, and more preferably 3 to 500 amino acids.

[0205] The above-mentioned protein or peptide includes, for example,interferons, interleukins, various growth factors such as fibroblastgrowth factor (aFGF, bFGF, etc.), (pro)urokinase, lymphotoxin, tumornecrosis factor (TNF), enzyme proteins such as β-galactosidase, storageproteins, streptoavisine, protein A, protein G, tissue plasminogenactivator (TPA), or muteins or parts (fragments) thereof.

[0206] Among those, fibroblast growth factor (aFGF, bFGF, etc.), or amutein thereof, or a part thereof (a fragment) (for example, bFGF CS23mutein, etc.) is preferably used.

[0207] An example of a bFGF CS23 mutein is a protein that comprises thefollowing amino acid sequence (SEQ ID NO. 13):Pro-Ala-Leu-Pro-Glu-Asp-Gly-Gly-Ser-Gly-Ala-Phe-Pro-Pro-Gly-His-Phe-Lys-Asp-Pro-Lys-Arg-Leu-Tyr-Cys-Lys-Asn-Gly-Gly-Phe-Phe-Leu-Arg-Ile-His-Pro-Asp-Gly-Arg-Val-Asp-Gly-Val-Arg-Glu-Lys-Ser-Asp-Pro-His-Ile-Lys-Leu-Gln-Leu-Gln-Ala-Glu-Glu-arg-Gly-Val-Val-Ser-Ile-Lys-Gly-Val-Ser-Ala-Asn-Arg-Tyr-Leu-Ala-Met-Lys-Glu-Asp-Gly-Arg-Leu-Leu-Ala-Ser-Lys-Ser-Val-Thr-Asp-Glu-Cys-Phe-Phe-Phe-Glu-Arg-Leu-Glu-Ser-Asn-Asn-Tyr-Asn-Thr-Tyr-Arg-Ser-Arg-Lys-Tyr-Thr-Ser-Trp-Tyr-Val-Ala-Leu-Lys-Arg-Thr-Gly-Gln-tyr-Lys-Leu-Gly-Ser-Lys-Thr-Gly-Pro-Gly-Gln-Lys-Ala-Ile-Leu-Phe-Leu-Pro-Met-Ser-Ala- Lys-Ser.

[0208] For the DNA encoding the fusion protein (including fusionpolypeptides) used in the method of the invention, (1) the entirenucleic acid sequence of said DNA may be chemically synthesized; or (2)said DNA may be constructed by adding the nucleic acid sequence encodingcysteine to the N-terminal of the nucleic acid sequence encoding aprotein, and then adding the nucleic acid sequence encoding thepolypeptide of the invention to the N-terminal. Moreover, (3) to obtaina fragment of the polypeptide of the invention, said DNA may beconstructed using a technique such as site-directed mutagenesis forsubstituting the amino acid residue just after the desired fragment withcysteine.

[0209] In the above producing method (1), well-known methods, forexample, phosphoamidide method, phosphotriester or diester method, orhydrogen phosphonate method may be used. The DNA of shorter length maybe synthesized completely at once, and the DNA of longer length may beproduced by synthesizing parts and ligating the parts using T4DNAligase.

[0210] In the above producing method (2), the DNA encoding the proteinon the C-terminal side is obtained by excising it from chromosomes witha suitable restriction enzyme and then subdloning it into a vector; orits cDNA is obtained. Subsequently, the DNA is cleaved with anappropriate restriction enzyme so that the N-terminal amino acid iscysteine, or a synthetic DNA is ligated to 5′-terminal of the DNAencoding the protein or a part thereof so that the N-terminal amino acidis cysteine after the modification. Finally, a DNA encoding theobjective protein (chemically synthesized, or cloned from an organism)is ligated to the 5′-terminal.

[0211] Examples of the DNA encoding the fusion protein obtained in theabove manner include a DNA represented by the following formula:AGCCTGAACTTTGAAGAACTGAAAGATTGGGGTCCGAAAAATGTGATTAAAATGAGCACCCCGGCGGTGAATAAAATGCCGCATAGCTTTGCGAATCTGC CGCTGCGTTTTTGC orTGT-R (1)

[0212] wherein, R indicates the following nucleic acid sequence (SEQ IDNO: 14, a fragment of bFGFCS23 mutein):CCCGAGGATGGCGGCAGCGGCGCCTTCCCGCCCGGCCACTTCAAGGACCCCAAGCGGCTGTACTGCAAAAACGGGGGCTTCTTCCTGCGCATCCACCCCGACGGCCGAGTTGACGGGGTCCGGGAGAAGAGCGACCCTCACATCAAGCTACAACTTCAAGCAGAAGAGAGAGGAGTTGTGTCTATCAAAGGAGTGAGCGCTAATCGTTACCTGGCTATGAAGGAAGATGGAAGATTACTAGCTTCTAAGTCTGTTACGGATGAGTGTTTCTTTTTTGAACGATTGGAATCTAATAACTACAATACTTACCGGTCAAGGAAATACACCAGTTGGTATGTGGCACTGAAACGAACTGGGCAGTATAAACTTGGATCCAAAACAGGACCTGGGCAGAAAGCTATACTTTTTCTTCCAATGTCTGCTAAGAGCTGC.

[0213] The above formula (I) means that the nucleic acid sequencerepresented by R is ligated to the DNA sequence encoding a polypeptidecomprising the amino acid sequence of SEQ ID NO: 7 through the nucleicacid sequence encoding cysteine.

[0214] The DNA (plasmid) having ATG on the 5′-terminal, a downstreamregion encoding the fusion protein and a translation termination codon,can be produced by chemical synthesis or by processing a cDNA encodingthe well-known protein, which is prepared by genetic engineering, or byprocessing a DNA encoding the protein, which is derived fromchromosomes.

[0215] In the present invention, the DNA encoding the fusion protein orpolypeptide, in which the polypeptide of the invention is attached tothe N-terminal of a protein or polypeptide having a cysteine at theN-terminal, can be modified by using conventional DNA technology; forexample, site-directed mutagenesis technology, so that it encodes thedesired mutein.

[0216] Site-directed mutagenesis technology is well known, and isdescribed in R. F. Lather and J. P. Lecoq: Genetic Engineering, AcademicPress (1983), p. 31-50. Oligonucleotide-directed mutagenesis isdescribed in M. Smith and S. Gillam: Genetic Engineering: Principles andMethods, Plenum Press (1981), Vol. 3, p. 1-32.

[0217] To produce a plasmid comprising a DNA region encoding the fusionprotein or polypeptide of the invention, in which the polypeptide of theinvention is linked to the N-terminal of a protein or polypeptide havinga cysteine at the N-terminal (hereinafter referred to as the fusionprotein of the invention), a plasmid vector derived from EscherichiaColi may be used: for example, pBR322 (Gene 2, 95(1977)), pBR313 (Gene2, 75(1977)), pBR324, pBR325 (Gene 4, 124(1978)), pBR327, pBR328 (Gene9, 287(1980)), pBR329 (Gene 17, 79(1982)), pKY2289 (Gene 3, 1(1978)),pKY2700 (Biochemistry 52, 770(1980)), pACYC177, pACYC184 (Journal ofBacteriology, 134, 1141 (1978)), pRK248, pRK646, pDF (Methods inEnzymology, 68, 268(1979)), pUC18, and pUC19 (Yanischperon, et al: Gene,33, 103(1985)). In addition, bacteriophage vectors may also be used: forexample, λ-phage vectors, e.g. λgt-vectors such as λgt-λC (Proc. Natl.Acad. Sci. U.S.A. 71, 4579(1974)), λgt-λB (Proc. Natl. Acad. Sci. U.S.A.72, 3461(1975)), λDam (Gene, 1, 255(1977)), and Charon vector (Science,196, 161(1977); Joural of Virology, 29, 555(1979)); and filamentousphage vectors, e.g. mp-vectors such as mp18, mp19 (Yanishperon et al,Gene, 33, 103(1985).

[0218] The said DNA preferably has a promoter upstream of the ATG, andsaid promoter may be any promoter suitable for the host used in theproduction of a transformant.

[0219] For example, for Escherichia coli, trp promoter, lac promoter,recA promoter, λPL promoter, lpp promoter, and T7 promoter, etc.; forBacillus subtilis, SP01 promoter, SP02 promoter, and penP promoter; forSaccharomyces cerevisiae, PHO5 promoter, PGK promoter, GAP promoter, andADH promoter; and for animal cells, a promoter derived from SV40, may beused. The SD (Shine and Delgamo) sequence may be inserted downstream ofthe promoter, if necessary.

[0220] When using T7 promoter system, any of 17 different T7 promoterslocated on T7 DNA (J. L. Oakley et al.,: Proc. Natl. Acad. Sci. U.S.A.,74: 4266-4270 (1977), M. D. Rosa, Cell, 16: 815-825 (1979), N.Panayotatos, et al., Nature, 280 : 35 (1979), J. J. Dunn et al., J. Mol.Biol., 166: 477-535 (1983)) may be used as the T7 promoter. φ10 promoter(A. H. Rosenberg et. al., Gene, 56: 125-135 (1987) is preferred.

[0221] As the transcription terminator, a terminator that functions inEscherichia coli strains, preferably Tφ terminator (F. W. Studier et.al., J. Mol. Biol., 189: 113-130 (1986)) may be used.

[0222] A gene of T7 RNA polymerase may be T7 Gene (F. W. Studier et.al., J. Mol. Biol., 189: 113-130 (1986)).

[0223] The preferred vector is one constructed by incorporating T7promoter and T7 terminator into the aforementioned vector. Such a vectorincludes pET-1, pET-2, pET-3, pET-4, pET-5 (A. H. Rosenberg, Gene 56:125-135 (1987), and pTB960-2 (EP-A-499990)). pTB960-2 is preferablyused.

[0224] The transformant of the invention can be produced by transforninga host with the expression plasmid obtained by the aforementionedmethod, according to a well-known method (e.g. S. N. Cohen, et al, Proc.Natl. Acad. Sci. U.S.A., 69, 2110 (1972)).

[0225] Microorganism hosts to be transformed include Escherichiabacteria, Bacillus bacteria, yeast, and animal cells.

[0226] Said Escherichia bacteria include Escherichia coli (E. coli),specifically, E.coli K12DH1 (Proc. Natl. Acad. Sci. U.S.A., 60, 160(1968)), JM-103 (Nucleic Acids Research, 9, 309 (1981)), JA221 (Journalof Molecular Biology, 120, 517 (1978)), HB101 (Journal of MolecularBiology, 41, 459 (1969)), C600 (Genetics, 39, 440 (1954)), N4830 (Cell,25, 713 (1981)), K-12 MM294 (Proc. Natl. Acad. Sci. U.S.A., 73, 4174(1976)), and BL-21.

[0227] Said Bacillus bacteria include Bacillus subtilis, specifically,Bacillus subtilis MI114 (Gene, 24, 255 (1983)), and 207-21 (Journal ofBiochemistry, 95, 87 (1984)).

[0228] Said yeast includes Saccharomyces cerevisiae, specifically,Saccharomyces cerevisiae AH22 (Proc. Natl. Acad. Sci. U.S.A., 75, 1929(1978)), XSB5 2-23C(Proc. Natl. Acad. Sci. U.S.A., 77, 2173 (1980)),BH-641A (ATCC 28339), 20B-12 (Genetics, 85, 23 (1976)), and GM3C-2(Proc. Natl. Acad. Sci. U.S.A., 78, 2258 (1981)).

[0229] Said animal cells includes Monkey cell COS-7 (Cell, 23, 175(1981)), Vero (Nihon Rinshou [Japan Clinic] 21, 1209 (1963)), Chinesehamster cells CHO (Journal of Experimental Medicine, 108, 945 (1985)),mouse L cells (Journal of National Cancer Institute, 4, 165 (1943)),human FL cells (Proceedings of the Society of Experimental Biology andMedicine, 94, 532 (1957)), and hamster C cells.

[0230] When utilizing T7 promoter system, an E.coli strain having T7 RNApolymerase gene (T7 gene 1) (F. W. Studier et al., Journal of MolecularBiology, 189, 113-130 (1986)) may be used as the host fortransformation, including E.coli MM294, DH-1, C600, JM109, BL21.Alternatively, an E.coli strain having T7 RNA polymerase gene (T7gene 1) in combination with another plasmid may also be used.Preferably, MM294 strain or BL21 strain is used, in which a λ phagehaving T7 gene 1 is lysogenized. In this case, the promoter for T7 gene1 may be lac promoter, under which the expression is induced byisopropyl-1-thio-β-D-galactopyranoside (IPTG).

[0231] The transformation of Bacillus bacteria hosts can be carried outaccording to a well-known method, such as one described in Molecular andGeneral Genetics, 168, 111 (1979).

[0232] The transformation of yeast hosts can be carried out according toa well-known method, such as one described in Proc. Natl. Acad. Sci.U.S.A. 75, 1929 (1978).

[0233] The transformation of animal cell hosts can be carried outaccording to a well-known method, such as one described in the Virology,52, 456 (1973).

[0234] The fusion protein of the invention can be produced by culturingthe above-described transformant in a medium, and collecting the fusionprotein produced.

[0235] It is desirable that the culture medium has pH of approximately 6to 8.

[0236] For example, M9 medium containing glucose and casamino acid(Journal of Experiments in Molecular Genetics, 431-433, Cold SpringHarbor Laboratory, New York, 1972) is preferable as the medium forculturing Escherichia bacteria. An agent such as 3β-indolyl acrylate orisopropyl β-D-thiogalactopyranside (IPTG) may be added to the medium inorder to increase the efficiency of the promoter, if necessary.

[0237] When the host is Escherichia bacteria, it is normally cultured atapproximately 15 to 43° C. for approximately 3 to 24 hours, and aerationand agitation may be applied if necessary.

[0238] When the host is Bacillus bacteria, it is normally cultured atapproximately 30 to 40° C. for approximately 6 to 24 hours, and aerationand agitation may be applied if necessary.

[0239] When culturing a yeast transformant, for example, Burkholderminimum medium (Bostian, K. L. et al., Proc. Natl. Acad. Sci. U.S.A.,77, 4505 (1980)) may be used. It is preferable to adjust the pH of theculture medium to approximately 5 to 8. Such a culture is normallyconducted at approximately 20 to 35° C. for approximately 24 to 72hours, and aeration and agitation may be applied if necessary.

[0240] When culturing an animal cell transformant, MEM medium (Science,122, 501 (1952)), DME medium (Virology, 8, 396 (1959)), RPMI 1640 mediun(The Journal of the American Medical Association, 199, 519 (1967)), and199 medium (Proceedings of the Society for Biological Medicine, 73, 1(1950)) containing approximately 0.2 to 20%, preferably approximately 5to 20% fetal bovine serum may be used. Tlhe pH is preferablyapproximately 6 to 8. Such a culture is normally conducted atapproximately 30 to 40° C. for approximately 15 to 60 hours, andaeration and agitation may be applied if necessary.

[0241] The fusion protein of the invention can be produced by culturingthe aforementioned transformant to express and accumulate the fusionprotein in the culture medium, and then harvesting it.

[0242] M9 medium including glucose and casamino acid (J. Miller,Experiments in Molecular Genetics, 431-433 (Cold Spring iHarborLaboratory, New York 1972)), 2× YT medium (Messing, Methods inEnzymology, 101, 20 (1983)), and LB medium may be used for the culturemedium.

[0243] Such a culture is normally conducted at approximately 15 to 43°C. for approximately 3 to 24 hours, and aeration and agitation may beapplied if necessary.

[0244] When using a recombinant cell having a λcI-ts repressor and anexpression vector having λPL-promoter, it is cultured at a temperatureof 15 to 36° C., preferably approximately 30 to 36° C., and it ispreferable to inactivate the λcI-ts repressor at approximately 37 to 42°C. In addition, in order to enhance the efficiency of the recA promoter,i.e. to reduce the function of inhibiting the recA gene expression,optionally, agents such as mitomycin C and nalidixic acid may be added,the culture may be exposed to ultraviolet light, or the pH of theculture medium may be changed to alkali side.

[0245] When using T7 promoter system, (1) IPTG, etc. is added to expressT7 gene (RNA polymerase gene) ligated downstream of the lac promoter; or(2) the temperature of the culture medium is increased to express T7gene (RNA polymerase gene) ligated downstream of the λPL-promoter. Asthe result, the expressed T7 phage RNA polymerase 1 acts on the T7promoter in a specific manner.

[0246] After the culture is finished, for example, the bacteria arecollected by a well-known method, suspended in a buffer solution, andthen crushed by treatment with a protein-denaturing agent,ultrasonication, enzymes such as lysozyme, glass beads, French press, orfreeze-thawing. The supernatant is obtained by a well-known method, suchas centrifugation.

[0247] Conventional protein purification methods may be employed toisolate the fusion protein from the supernatant obtained above. Anappropriate combination of, for example, gel filteration, ion-exchangechromatography, absorption chromatography, high speed liquidchromatography, affinity chromatography, hydrophobic chromatography, andelectrophoresis may be used.

[0248] When the N-terminal of the polypeptide of the invention isIsoleucine, Valine, Cysteine, Threonine, Aspartic acid, Lysine, Leucine,Arginine, Asparagine, Methionille, Phenylalanine, Tyrosine, Tryptophan,Glutamic acid, Glutamine or Histidine (for example, when the polypeptideis one having the amino acid sequence shown in SEQ ID NO: 3, 5, 9 or11), there are cases in which methionine derived from the translationinitiation codon is added to the N-terminal of the fusion proteinobtained as described above.

[0249] Alternatively, the fusion protein may be submitted to thefollowing reaction step without being purified or in a partiallypurified state.

[0250] Subsequently, the fusion protein of the invention thus obtainedis submitted to a reaction for cleavage of the peptide bond on theamino-terminal side of the cysteine residue.

[0251] This cleavage reaction may be, for example, S-cyanation reaction,which is followed by a hydrolysis reaction.

[0252] When an amide form of the polypeptide or a salt thereof isdesired as the final product, the S-cyanation reaction is followed byammonolysis for the cleavage reaction.

[0253] The S-cyanation reaction is conducted by reacting an S-cyanationreagent with the source material.

[0254] The S-cyanation reagent includes 2-nitro-5-thiocyanobenzoic acid(NTCB), 1-cyano-4-dimethyl aminopyridinium salt (DMAP-CN), and CN⁻ ions.The used amount of the S-cyanation reagents may be approximately 2 to 50times to the total amount of thiol group, and preferably approximately 5to 10 times.

[0255] The reaction temperature may be between approximately 0 and 80°C., and preferably between approximately 0 and 50° C. Any buffersolution may be used as the solvent as long as it does not react withthe S-cyanation reagent, including tris-hydrochloride buffer solution,tris-acetate buffer solution, phosphate buffer solution, and boratebuffer solution. An organic solvent may be further contained as long asit does not react with the S-cyanation reagent.

[0256] The cleavage reaction may be carried out at pH of 1 to 12. Inparticular, pH 7 to 10 is preferable when using NTCB, and pH 2 to 7 ispreferable when using DMAP-CN in order to prevent an S—S exchangereaction. The reaction solution may further contain a denaturing agentsuch as guanidine hydrochloride.

[0257] Alkali treatment may be carried out for the aforementionedhydrolysis reaction.

[0258] The alkali treatment is conducted by adjusting the pH of anaqueous solution containing the source material to 7 to 14.

[0259] For the pH adjustment, a suitable amount of a solution ofammonia, sodium hydroxide, amino compound, trizma base(tris(hydroxymethyl)-aminomethane), sodium II phosphate, potassiumhydroxide, or barium hydroxide is added to the aqueous solutioncontaining the source material. In particular, ammonia solution ispreferable.

[0260] For the aforementioned treatment, the following concentration ofeach solution may be used: approximately 0.01 to 15 N, preferablyapproximately 0.1 to 5 N, for ammonia or amino compound; approximately0.01 to 2 N, preferably approximately 0.05 to 1 N, for sodium hydroxide;approximately 1 mM to 1 M, preferably approximately 20 mM to 200 mM, fortrizma base; approximately 1 mM to 1 M, preferably approximately 10 mMto 100 mM, for sodium II phosphate; approximately 0.01 to 4 N,preferably approximately 0.1 to 2 N, for potassium hydroxide;approximately 0.01 to 0.2 M, preferably approximately 0.1 to 0.2 M, forpotassium hydroxide. The reaction temperature may be betweenapproximately −20 and 80° C., and preferably between approximately −10and 50° C.

[0261] The reaction time for S-cyanation reaction may be approximately 1to 60 min., preferably approximately 15 to 30 min.; for ammonolysis,approximately 5 min. to 24 hours, preferably approximately 10 to 180min.; and for hydrolysis reaction, approximately 5 min. to 100 hours,preferably approximately 10 min. to 15 hours.

[0262] The S-cyanation reaction or the hydrolysis reaction may occur asindicated in FIG. 1. In FIG. 1, X represents R¹—(NR²)— or OH, wherein R¹and R² may be the same or different, and each indicates (i) hydrogen;(ii) C₁₋₂₀ alkyl, C₃₋₈ cycloalkyl, C₆₋₁₄ aryl, or C₆₋₁₄ aryl-C₁₋₃ alkyl(which may be unsubstituted or substituted with 1 to 3 amino groups orhydroxyl groups on a carbon atom); (iii) amino optionally substituted;(iv) hydroxyl or C₁₋₆ alkoxy.

[0263] The aforementioned C₁₋₂₀ alkyl indicates, for examples, methyl,ethyl, propyl, isopropyl, butyl, sec-butyl, penthyl, isopenthyl,neopenthyl, 1-ethylpenthyl, hexyl, isohexyl, heptyl, octyl, nonanyl,decanyl, undecanyl, dodecanyl, tetradecanyl, pentadecanyl, hexadecanyl,heptadecanyl, octadecanyl, nonadecanyl, and eicosanyl.

[0264] The aforementioned C₃₋₈ cycloalkyl indicates, for examples,cyclopropyl, cyclobutyl, cyclopenthyl, cyclohexyl, cycloheptyl, andcyclooctyl.

[0265] The aforementioned C₆₋₁₄ aryl indicates, for examples, phenyl,naphthlyl, anthryl, phenanthryl, and acenaphthylenyl.

[0266] The aforementioned C₆₋₁₄ aryl-C₁₋₃-alkyl indicates, for examples,benzyl, phenethyl, 3-phenylpropyl, (1-naphthyl)methyl, and(2-naphthyl)methyl.

[0267] The aforementioned C₁₋₆ alkoxy indicates, for examples, methoxy,ethoxy, propoxy, butoxy, penthyloxy, and hexyloxy.

[0268] Substituents for the amino group optionally substituted,described in (iii) above, include amino acids, and peptides having 2 to10 amino acids.

[0269] The above amino acids may be L-forn or D-form, including Ala,Arg, Asp, Asn, Glu, Gln, Gly, His, lle, Met, Leu, Phe, Pro, Ser, Thr,Trp, Tyr, and Val.

[0270] When using ammonia or an amino compound in the reaction, thecorresponding amide form is obtained.

[0271] In general, known methods for peptide purification may be used toisolate the excised polypeptide of the invention. An appropriatecombination of, for example, gel filtering, ion-exchange chromatography,high speed liquid chromatography, affinity chromatography, hydrophobicchromatography, thin layer chromatography or electrophoresis may beused.

[0272] As described above, there is a case where a methionine derivedfrom the translation initiation codon is added to the N-terminal of thepolypeptide of the invention. In this case, it is possible to remove theN-terminal methionine, for example, by a reaction with an α-diketonesuch as glyoxylic acid (preferably, in the presence of a transitionmetal ion such as copper sulfate, and a base such as pyridine); and asubsequent hydrolysis with a diamine such as o-phenylenediamine. Such amethod is described in detail below.

[0273] [The method of removing the N-terminal methionine from thepolypeptide of the invention]

[0274] In the specification, the “methionine residue optionallyoxidized” indicates a methionine residue or its S-oxidized form. TheS-oxidized form of methionine residue includes a sulfoxide or sulfonethereof.

[0275] The polypeptide of the invention having the methionine residueoptionally oxidized at the N-terminal includes a polypeptide representedby:

[0276] the formnula CH₃—S(O)_(m)—(CH₂)₂—CO—X

[0277] wherein m indicates an integer of 0 to 2, and X indicates apeptide chain of the polypeptide of the invention.

[0278] The polypeptide may be in a salt form, including the same saltforms as those of the polypeptide of the invention as described above.

[0279] The polypeptide of the invention having the methionine residueoptionally oxidized at the N-terminal, or a salt thereof is preferablyone produced by the genetic engineering.

[0280] In the said formula, m is preferably 0. The peptide chain of thepolypeptide of the invention as indicated by X includes the polypeptideof the invention as described above.

[0281] The said polypeptide of the invention or a salt thereof may berefolded (activated, renatured) after or before being subjected to theprocess of removing the N-terminal methionine residue optionallyoxidized or a diketone of the methionine residue.

[0282] In the specification, the polypeptide of the invention having adiketone of the methionine residue optionally oxidized at theN-terminal, or a salt thereof indicates a compound represented by:

[0283] the formula CH₃—S(O)_(m)—(CH₂)₂—CO—CO—X

[0284] wherein m indicates an integer of 0 to 2, and X indicates apeptide chain of the polypeptide of the invention, or a salt thereof.The polypeptide of the invention having a diketone of the methionineresidue optionally oxidized at the N-terminal, or a salt thereof can beproduced through a variety of reactions such as a chemical reaction andan enzyme reaction. For example, the polypeptide of the invention havinga diketone of the methionine residue optionally oxidized at theN-terminal, or a salt thereof can be produced by a transaminationreaction, in which the polypeptide of the invention having a methionineresidue optionally oxidized at the N-terminal, or a salt thereof isreacted with an α-diketone.

[0285] In the specification, the α-diketone may be any one capable ofcarrying out the transamination of the said polypeptide of the inventionor a salt thereof, and includes a compound represented by:

[0286] the formula R1-CO—CO—R2

[0287] wherein R1 indicates a hydrogen, or a lower alkyl or phenylgroup, which may be optionally substituted with a carboxyl group(preferably hydrogen or methyl; more preferably hydrogen); and R2indicates a hydroxyl, a lower alkoxy, or an amino group optionallysubstituted with a lower alkyl (preferably hydroxy), or a salt thereof.

[0288] In the said formula, the lower alkyl group represented by R1includes a C₁₋₆-alkyl such as methyl, ethyl, propyl, i-propyl, butyl,i-butyl, sec-butyl, t-butyl. The lower alkoxy group represented by R2includes a C₁₋₆-alkoxy such as methoxy, ethoxy, propoxy, i-propoxy,butoxy, i-butoxy, sec-butoxy, t-butoxy. The amino group optionallysubstituted with a lower alkyl indicates an amino group, which may haveone or two lower alkyl groups as represented by R1. In addition, thesaid salt form includes the same salt forms as described regarding thesalts of the polypeptide of the invention having the methionine residueoptionally oxidized at the N-terminal.

[0289] Examples of the α-diketone include glyoxylic acid, pyruvic acid,oxalacetic acid, phenylglyoxylic acid, and 2-oxoglutaric acid. Amongthem, glyoxylic acid is preferably used.

[0290] The α-diketone may be in a salt form, including an alkali metalsalt such as a sodium salt, a potassium salt; and a salt with aninorganic acid such as a hydrochloride.

[0291] In the transamination reaction, it is generally preferred thatthe polypeptide of the invention having a methionine residue optionallyoxidized at the N-terminal, or a salt thereof is reacted with anα-diketone in an amount of about 1 to 10000 moles (preferably 2000 to4000 moles) of the α-diketone per mole of the polypeptide of theinvention or a salt thereof at a temperature between 0 and 70° C.(preferably between about 20 and 40° C.) for a time period between about10 min and 5 hr (preferably between about 20 min and 2 hr). Any buffersolution may be used, which does not inhibit the transamination,including phosphate buffer, acetate buffer, and citrate buffer. Inparticular, an acetate buffer solution is preferred. The pH of thereaction solution is adjusted to about 2 to 9, preferably about 4 to 7,in particular, about 5 to 6 to achieve the reaction.

[0292] It is preferred that the reaction with an α-diketone is carriedout in the presence of a transition metal ion to enhance thetransamination. Generally, 0.001 to 0.1 mole (preferably 0.01 to 0.05mole) of the transition metal ion per mole of α-diketone is used.Examples of the transition metal ion include a copper ion (Cu+, Cu2+),cobalt ion (Co2+, Co3+), nickel ion (Ni2+, Ni3+), iron ion (Fe2+, Fe3+),zinc ion (Zn2+), aluminum ion (Al3+), manganese ion (Mn2+, etc), galliumion (Ga3+), indium ion (In3+), magnesium ion (Mg2+), and calcium ion(Ca2+). Among these, preferably copper ion and cobalt ion; morepreferably copper ion is used. The transition metal ion may be added tothe reaction medium in a salt form with an inorganic acid, such assulfuric acid, nitric acid, hydrochloric acid, or perchloric acid, or ina salt form with an organic acid, such as acetic acid, oxalic acid,citric acid, or carbonic acid. Among these, preferably copper sulfateand copper acetate; more preferably copper sulfate is used.

[0293] In addition, it is preferred that the reaction with an α-diketoneis carried out in the presence of a base. Generally, 0.1 to 20 moles(preferably 0.5 to 10 moles) of the base per mole of α-diketone areused. Examples of the base include an alkyl amine such as triethylamine,tributylamine, and an aromatic amine such as N,N-dimethylaniline,pyridine, lutidine, collidine, 4-(dimethylamino) pyridine, andimidazole. Among these, pyridine is preferably used.

[0294] In the transamination reaction, it is preferred that urea isfurther contained in the buffer solution for the transamination in orderto prevent the precipitation of the polypeptide of the invention havinga methionine residue optionally oxidized at the N-terminal or a saltthereof, and also the polypeptide of the invention having a diketone ofthe methionine residue optionally oxidized at the N-terminal or a saltthereof, which is produced by the transamination of the polypeptide ofthe invention having the methionine residue. For example, urea is addedto the buffer solution preferably at a final concentration of about 1 to8 M, more preferably about 3 to 6 M.

[0295] Furthermore, in the transamination reaction, it is preferred thatthe reaction with an α-diketone is carried out in the presence of atransition metal ion and a base. In practice, a mixture containing thethree components, i.e. a transition metal ion, a base and an α-diketone(for example, copper sulfate, pyridine and glyoxylic acid) is added tothe aqueous solution containing the polypeptide of the invention havinga methionine residue optionally oxidized at the N-terminal or a saltthereof to achieve the transamination.

[0296] The compound or a salt thereof, produced by the transaminationreaction and represented by:

[0297] the formula CH₃—S(O)_(m)—(CH₂)₂—CO—CO—X

[0298] wherein m indicates an integer of 0 to 2, and X indicates apeptide chain of the polypeptide of the invention, (i.e. the polypeptideof the invention having a diketone of the methionine residue optionallyoxidized at the N-terminal or a salt thereof) can be isolated andpurified from the reaction solution by well-known peptide/proteinpurification techniques, for example, extraction, salting out,distribution, re-crystallization, or chromatography, before it issubjected to a hydrolysis reaction. Alternatively, the compound may besubjected to the hydrolysis reaction as it is without being purified.

[0299] The polypeptide of the invention having a diketone of themethionine residue optionally oxidized at the N-terminal or a saltthereof, which is produced by the transamination, can be generallysubjected to the hydrolysis reaction with a base, so that it can bechanged to the polypeptide of the invention not having the methionineresidue optionally oxidized or the diketone thereof at the N-terminal,or a salt thereof.

[0300] Examples of the base used in the hydrolysis reaction include anamine and a salt thereof, for example, an alkyl amine such ascysteamine, triethylamine, tributylamine, and salts thereof; an aromaticamine such as N,N-dimethylaniline, pyridine, lutidine, collidine,4-(dimethylamino) pyridine, imidazole, and salts thereof; a diamine suchas o-phenylenediamine, tolylene-3,4-diamine, 3,4-diamino-benzoic acid,N-alkyl-substituted forms thereof (e.g. N-methyl-1,2-phenylenediamine,N-ethyl-1,2-phenylenediamine, N-isopropyl-1,2-phenylenediamine),2,3-diaminophenol, 4-chloro-o-phenylenediamine (preferably an aromaticdiamine, especially 3,4-diamino-benzoic acid and N-alkyl-substitutedforms thereof (e.g. N-methyl-1,2-phenylenediamine,N-ethyl-1,2-phenylenediamine, N-isopropyl-1,2-phenylenediamine)), andsalts thereof; a thiosemicarbazide such as thiosemicarbazide, acetonethiosemicarbazide, phenyl thiosemicarbazide; and a selenosemicarbazidesuch as selenosemicarbazide, acetone selenosemicarbazide. Among these,an amine, especially a diamine or a thiosemicarbazide, or a salt thereofis preferably used. In particular, 3,4-diamino-benzoic acid and a saltthereof are preferred.

[0301] The salt of the base used in the hydrolysis reaction includes thesame salt forms as described regarding the salts of the polypeptide ofthe invention having the methionine residue optionally oxidized at theN-terminal.

[0302] The amount of the base is usually about 1 to 10000 moles,preferably about 200 to 3000 moles, and more preferably about 500 to3000 moles per mole of the polypeptide of the invention having thediketone of the methionine residue or a salt thereof. The hydrolysisreaction is usually carried out at a temperature between 0 and 70° C.(preferably between about 20 and 40° C.) for a time period between about1 hr and 7 days (preferably between about 10 hrs and 5 days). A buffersolution is preferably used as a medium in the reaction, includingphosphate buffer, acetate buffer, citrate buffer, and formate buffer.Any buffer solution may be used, which does not inhibit the hydrolysisreaction. In particular, preferred are the formate buffers, such asacetic acid/sodium formate buffer, formic acid/sodium formate buffer,and formic acid/sodium acetate buffer. The pH of the reaction solutionis adjusted to about 2 to 9, preferably about 3 to 7, in particular,about 4 to 6 to achieve the reaction. Such a buffer is preferably usedat a concentration of about 0.1 to 8 mol/L, more preferably about 0.5 to6 mol/L.

[0303] In the hydrolysis reaction, it is preferred that urea is furthercontained in the buffer solution for the hydrolysis in order to preventthe precipitation of the polypeptide of the invention having a diketoneof the methionine residue optionally oxidized at the N-terminal or asalt thereof, and also the polypeptide of the invention not having themethionine residue or a salt thereof, which is produced by thehydrolysis reaction. For example, urea is added to the buffer solutionpreferably at a final concentration of about 1 to 6 M, more preferablyabout 2 to 5 M.

[0304] The polypeptide or its salt of the invention obtained in this waymay also be isolated and purified from the reaction solution bywell-known purification techniques, for example, extraction, saltingout, distribution, re-crystallization, or chromatography. The preferablepurification method is ion-exchange chromatography using SP-sepharose(Pharmacia Biotech, Co., Ltd.) or DEAE-5PW (Tosoh Co., Ltd.).

[0305] The polypeptide or its salt of the invention produced by themethod of the invention may be formulated with sterilized water, humanserum albumin (HSA), physiological saline and other well-knownphysiologically acceptable carriers, and then may be administered to amammal (e.g. human) parenterally or locally. For example, thepolypeptide or its salt of the invention may be administeredparenterally by intravenous or intramuscular injection in a daily doseper person of approximately 0.01 to 50 mg, preferably approximately 0.1to 10 mg.

[0306] The formulation comprising the polypeptide or its salt of theinvention produced by the method of the invention may further containsalts, diluents, adjuvants, other carriers, buffers, binders,surfactants, preservatives, and other physiologically acceptable activecomponents. The parenteral dosage formulation may be provided as anampoule of a suspension in a sterilized water solution orphysiologically acceptable solvent, or as an ampoule of sterilizedpowder (normally obtained by lyophilization of the peptide solution)which can be diluted with a physiologically acceptable diluent at use.

[0307] The polypeptide of the invention produced by the method of theinvention has an activity of regulating, i.e. promoting or inhibitingthe prolactin secretion. On one hand, the polypeptide of the inventionhas an activity of promoting the prolactin secretion, and is thus usefulas a prophylactic or therapeutic agent for various diseases associatedwith prolactin hypo-secretioni. On the other hand, the polypeptide ofthe invention also shows an activity of inhibiting the prolactinsecretion, which is mediated by the induction of desensitization of theprolactin secretion by administrating an increasing amount of thepolypeptide having a strong affinity to its receptor protein. In thiscase, the polypeptide of the invention can be used as a prophylactic ortherapeutic agent for various diseases associated with prolactinhyper-secretion.

[0308] Accordingly, the polypeptide of the invention is useful as anenhancer of the prolactin secretion or a prophylactic or therapeuticagent for various diseases relating to the prolactin secretion, e.g.hypoovarianism, seminal gland hypoplasia, osteoporosis, menopausaldisorder, lactation failure, hypothroidism, renal failure.

[0309] In addition, the polypeptide of the invention is useful as anaphrodisiac because it has an aphrodisiac effect (pheromonal effect)based on its activity of inhibiting the prolactin secretion.

[0310] Meanwhile, the polypeptide of the invention is useful as aninhibitor of the prolactin secretion or a prophylactic or therapeuticagent for various diseases relating to the prolactin secretion, e.g.hyperprolactinemia, pituitary gland tumor, diencephalic tumor, menstrualdisorder, stress, autoimmmnune disease, prolactinoma, infertility,impotence, amenorrhea, chylorrhea, acromegaly, Chiari-Frommel syndrome,Argonz-del Castilo syndrome, Forbes-Albright syndrome, breast carcinoma,lymphoma, Sheehan syndrome, or dysspermatogenesis.

[0311] In addition, the polypeptide of the invention is also useful asan anticonceptive, due to its activity of inhibiting the prolactinsecretion.

[0312] Further, the polypeptide of the invention is also useful as anassay agent to examine the prolactin secretion activity, and as ananimal drug, for example, a lactation enhancer in domestic mammals suchas a cow, goat and pig, which is expected to be applied, for example, inthe production of a utile material, where the utile material may beproduced inside such a domestic mammal, and released into milk thereof.

[0313] Furthermore, the polypeptide of the invention has the activity ofregulating placental function, and thus is also useful as a prophylacticor therapeutic agent for choriocarcinoma, hydatid mole, invasive mole,miscarriage, fetal dysgenesis, dysbolism of saccharide, dysbolism oflipid, or induction of delivery.

[0314] In the present specification and drawings, codes of amino acids,peptides, protecting groups, active groups, and other substances aredenoted on the base of the IUPAC-IUB (Commission on BiochemicalNomenclature) or conventional codes used in the related field. Examplesare shown below. In addition, when an amino acid can have opticalisomers, unless specifically noted, the L-form is meant. DNADeoxyribonucleic acid A Adenine T Thymine G Guanine C Cytosine RNARibonucleic acid EDTA Ethylene diamine tetra acetic acid Gly Glycine AlaAlanine Val Valine Leu Leucine Ile Isoleucine Ser Serine Thr ThreonineMet Methionine Glu Glutamic acid Asp Aspartic acid Lys Lysine ArgArginine His Histidine Phe Phenylalanine Tyr Tyrosine Trp Tryptophan ProProline Asn Asparagine Gln Glutamine ATP Adenosine triphospate

[0315] The SEQ ID NOs. in the Sequence Listing indicate the followingsequences.

[0316] [SEQ ID NO: 1]

[0317] This sequence shows the amino acid sequence of the polypeptide ofthe invention consisting of 9 amino acid residues.

[0318] [SEQ ID NO: 2]

[0319] This sequence shows the base sequence of the DNA encoding thepolypeptide having the amino acid sequence of SEQ ID NO: 1.

[0320] [SEQ ID NO: 3]

[0321] This sequence shows the amino acid sequence of the polypeptide ofthe invention consisting of 12 amino acid residues.

[0322] [SEQ ID NO: 4]

[0323] This sequence shows the base sequence of the DNA encoding thepolypeptide having the amino acid sequence of SEQ ID NO: 3.

[0324] [SEQ ID NO: 5]

[0325] This sequence shows the amino acid sequence of the polypeptide ofthe invention consisting of 20 amino acid residues.

[0326] [SEQ ID NO: 6]

[0327] This sequence shows the base sequence of the DNA encoding thepolypeptide having the amino acid sequence of SEQ ID NO: 5.

[0328] [SEQ ID NO: 7]

[0329] This sequence shows the amino acid sequence of the polypeptide ofthe invention consisting of 37 amino acid residues.

[0330] [SEQ ID NO: 8]

[0331] This sequence shows the base sequence of the DNA encoding thepolypeptide having the amino acid sequence of SEQ ID NO: 7.

[0332] [SEQ ID NO: 9]

[0333] This sequence shows the amino acid sequence of the polypeptide ofthe invention consisting of 9 amino acid residues.

[0334] [SEQ ID NO: 10]

[0335] This sequence shows the base sequence of the DNA encoding thepolypeptide having the amino acid sequence of SEQ ID NO: 9.

[0336] [SEQ ID NO: 11]

[0337] This sequence shows the amino acid sequence of the polypeptide ofthe invention consisting of 17 amino acid residues.

[0338] [SEQ ID NO: 12]

[0339] This sequence shows the base sequence of the DNA encoding thepolypeptide having the amino acid sequence of SEQ ID NO: 11.

[0340] [SEQ ID NO: 13]

[0341] This sequence shows the amino acid sequence of bFGF CS23 mutein.

[0342] [SEQ ID NO: 14]

[0343] This sequence shows the base sequence of the gene fragmentencoding the fusion protein represented by formula (I) .

[0344] [SEQ ID NO: 15]

[0345] This sequence shows the base sequence of primer # 1 used forpreparing the structural gene of the polypeptide of the invention inExample 1.

[0346] [SEQ ID NO: 16]

[0347] This sequence shows the base sequence of primer # 2 used forpreparing the structural gene of the polypeptide of the invention inExample 1.

[0348] [SEQ ID NO: 17]

[0349] This sequence shows the base sequence of primer # 3 used forpreparing the structural gene of the polypeptide of the invention inExample 1.

[0350] [SEQ ID NO: 18]

[0351] This sequence shows the base sequence of primer # 4 used forpreparing the structural gene of the polypeptide of the invention inExample 1.

[0352] [SEQ ID NO: 19]

[0353] This sequence shows the amino acid sequence of the humanprecursor polypeptide of the invention.

[0354] [SEQ ID NO: 20]

[0355] This sequence shows the base sequence of the DNA encoding theprecursor polypeptide having the amino acid sequence of SEQ ID NO: 19.

[0356] [SEQ ID NO: 21]

[0357] This sequence shows the amino acid sequence of the humanprecursor polypeptide of the invention.

[0358] [SEQ ID NO: 22]

[0359] This sequence shows the base sequence of the DNA encoding theprecursor polypeptide having the amino acid sequence of SEQ ID NO: 21.

[0360] [SEQ ID NO: 23]

[0361] This sequence shows the amino acid sequence of the bovineprecursor polypeptide of the invention.

[0362] [SEQ ID NO: 24]

[0363] This sequence shows the base sequence of the DNA encoding theprecursor polypeptide having the amino acid sequence of SEQ ID NO: 23.

[0364] [SEQ ID NO: 25]

[0365] This sequence shows the amino acid sequence of the mouseprecursor polypeptide of the invention.

[0366] [SEQ ID NO: 26]

[0367] This sequence shows the base sequence of the DNA encoding theprecursor polypeptide having the amino acid sequence of SEQ ID NO: 25.

[0368] [SEQ ID NO: 27]

[0369] This sequence shows the amino acid sequence of the rat precursorpolypeptide of the invention.

[0370] [SEQ ID NO: 28]

[0371] This sequence shows the base sequence of the DNA encoding theprecursor polypeptide having the amino acid sequence of SEQ ID NO: 27.

[0372] [SEQ ID NO: 29]

[0373] This sequence shows the amino acid sequence of the rat precursorpolypeptide of the invention.

[0374] [SEQ ID NO: 30]

[0375] This sequence shows the base sequence of the DNA encoding theprecursor polypeptide having the amino acid sequence of SEQ ID NO: 29.

[0376] The transformant Escherichia coli MM294 (DE3)/pTFCRFRP-1,obtained in Example 2 described later, is deposited in the InternationalPatent Organism Depositary, the National Institute of AdvancedIndustrial Science and Technology (now-defunct National Institute ofBioscience and Human Technology (NIBH), Agency of Industrial Science andTechnology, Ministry of International Trade and Industry), located atCenter No. 6, 1-1-1 Higasi, Tukuba-shi, Ibaraki, 305-8566, Japan, underAccession Number FERM BP-7313 as of Sep. 28, 2000; and in the Institutefor Fermentation, Osaka (IFO), located at 2-17-85, Jyuso-Honmati,Yodogawa-ku, Osaka-shi, Osaka, 532-8686, Japan, under Accession NumberIFO 16476 since Sep. 19, 2000.

[0377] The present invention will be described in more detail below withreference to the following examples, which are however not intended tolimit the scope of the present invention thereto.

EXAMPLES Example 1

[0378] Preparation of the Structural Gene Encoding the Polypeptide ofthe Invention (the Polypeptide Having the Amino Acid Sequence of SEQ IDNO: 7)

[0379] The structural gene encoding the polypeptide of the invention(the polypeptide having the amino acid sequence of SEQ ID NO: 7) wasprepared using 4 different DNA fragments represented by SEQ ID NOs: 15to 18 (# 1: SEQ ID NO: 15; # 4: SEQ ID NO: 18, Kikotek Co.)(# 2: SEQ IDNO: 16; # 3: SEQ ID NO: 17, Amersham Pharmacia Biotech Co.) according toa well-known method.

[0380] (a) Phosphorylation of the DNA Oligomers

[0381] The phosphorylation of 5′ terminals of the 2 oligomers, excluding# 1 and # 4, was conducted by incubating 1 μg of each DNA oligomer at37° C. for one hour in 100 μL phosphorylation reaction solution (50 mMTris-HCl (pH 7.6), 10 mM MgCl₂, 1 mM spermidine, 10 mM dithiothreitol,0.1 mg/mL bovine serum albumin, 1 mM ATP, and 10 units T4 polynucleotidekinase (Nippon Gene)). After treating the solution with phenol, 2 timesvol. of ethanol was added to the aqueous layer recovered. After cooledto −70° C., the DNA was precipitated by centrifugation.

[0382] (b) Ligation of the DNA Fragments

[0383] The DNA fragments phosphorylated in (a), # 1 fragment, and # 4fragment were mixed and added to the solution of 10 mM Tris/HCl and 2 mMEDTA (pH8.0) in a final volume of 120 μl. This mixture was incubated 80°C. for 10 min, and then cooled slowly to a room temperature to achieveannealing. The ligation was conducted using TaKaRa DNA Ligation Kit ver.2 (Takara) by adding 30 μL of the Kit solution II to 30 μL of theannealing solution, mixing well, adding 60 μL of the Kit solution I, andthen incubating at 37° C. for one hour. After treating the solution withphenol, 2 times vol. of ethanol was added to the aqueous layerrecovered. After cooled to −70° C., the DNA was precipitated bycentrifugation.

[0384] (c) Phosphorylation of the 5′-Terminal

[0385] The precipitated DNA was dissolved in 10 μL of TE buffer solution(10 mM Tris-HCl (pH8.0), 1 mM EDTA), and incubated at 37° C. for onehour in 100 μL phosphorylation reaction solution (50 mM Tris-HCl (pH7.6), 10 mM MgCl₂, 1 mM spermidine, 10 mM dithiothreitol, 0.1 mg/mLbovine serum albumin, 1 mM ATP, and 10 units T4 polynucleotide kinase(Nippon Gene)) for the phosphorylation of the 5′-terminal. Aftertreating the solution with phenol, 2 times vol. of ethanol was added tothe aqueous layer recovered. After cooled to −70° C., the DNA wasprecipitated by centrifugation, and then dissolved in 20 μL of TE buffersolution.

Example 2

[0386] Preparation of the Expression Plasmid for the Polypeptide of theInvention (the Polypeptide Having the Amino Acid Sequence of SEQ ID NO:7)

[0387] To make the expression vector, pTFC (Publication of PatentApplication: No.2000-270871) was digested with Ndel and Aval (Takara) at37° C. for 4 hours. After 1% agarose gel electrophoresis, 4.4 kb DNAfragment was extracted using the QIAquick Gel Extraction Kit (QIAGEN)and was dissolved in 25 μL TE buffer solution. This Ndel/Aval fragmentderived from pTFC and the structural gene of the polypeptide (having theamino acid sequence of SEQ ID NO: 7) as prepared above were ligatedusing the TaKaRa DNA Ligation Kit ver. 2 (Takara).

[0388]Escherichia coli JM109 competent cells (Toyobo) were transformedwith 10 μL of the ligation solution, inoculated on an LB agar mediumcontaining 10 μg/mL tetracycline. After cultured overnight at 37° C., atetracycline resistant colony was selected. The transformant wascultured overnight at 37° C. in LB medium, and the plasmid pTFCRFRP-1was prepared from the culture using the QIAprep8 Miniprep Kit(QIAGEN)(FIG. 2). The nucleic acid sequence of the structural gene ofthe polypeptide (having the amino acid sequence of SEQ ID NO: 7) wasconfirmed using the Applied Biosystems, Model 377 DNA sequencer.Escherichia coli MM294 (DE3) was transformed with the plasmid pTFCRFRP-1to obtain a strain of Escherichia coli, M294(DE3)/pTFCRFRP-1 expressingRFRP-1/CS23 fusion protein.

Example 3

[0389] The transformant obtained in Example 2 was cultured with shakingat 37° C. for 8 hours in a 2-L flask containing 1 L of LB medium (1%peptone, 0.5% yeast extract, 0.5% sodium chloride) containing 5.0 mg/Ltetracycline. The culture obtained was transferred to a 50-L fermentorcontaining 19 L of a main fermentation medium (1.68% sodium monohydrogenphosphate, 0.3% potassium dihydrogen phosphate, 0.1% ammonium chloride,0.05% sodium chloride, 0.05% magnesium sulfate, 0.02% defoaming agent,0.00025% ferrous sulfate, 0.00025% thiamin hydrochloride, 1.5% glucose,and 1.5% casamino acid), and then incubated at 30° C. with aeration andagitation. At 500 kleets of the turbidity in the culture,isopropyl-β-D-thiogalactopyranoside was added to the culture at a finalconcentration of 12 mg/L, and the culture was incubated for another 6hours. After the culture was completed, approximately 500 g (wet weight)cells were obtained by centrifugation of the culture, and stored in afrozen state at −80° C.

Example 4

[0390] Production of the Polypeptide of the Invention (the PolypeptideHaving the Amino Acid Sequence of SEQ ID NO: 7)

[0391] To 500 g of the cells obtained in Example 2, 1000 mL of thesolution of 6 M guanidine hydrochloride and 0.2 M Tris/HCl (pH 8.0) wasadded. After shaking for about 4 hrs., the centrifugal separation wasconducted (10,000 rpm, 60 min.) to obtain a supernatant, which wasdiluted with 29 L of 0.6 M arginine, 1 mM dithiothreitol, and 50 mMTris/HCl (pH 8.0). After an overnight incubation at 10° C., thesupernatant was adjusted to pH 6.0, and passed through an AF-HeparinToyopearl 650M column (11.3 cm ID×13.5 cm L, Tosoh) for the absorption,which had been equilibrated with 50 mM phosphate buffer solution (pH6.0). After the column was washed, the elution was made with 50 mMphosphate buffer solution containing 2M NaCl, pH 6.0 to obtain 1000 μLof the fraction containing the polypeptide (having the amino acidsequence of SEQ ID NO: 7)/CS23 fusion protein.

[0392] This effluent was conceintrated using the Pellicon Mini Cassette(Millipore) with 0.1 M acetic acid added thereto to obtain thepolypeptide (having the amino acid sequence of SEQ ID NO: 7)/CS23 fusion.pyotein in 0.1 M acetic acid solution. After adding urea to thesolution at a final concentration of 6 M, 445 mg of1-cyano-4-diethylaminopyridinium salt (DMAP-CN) was added and incubatedfor 15 min. at room temperature for the reaction. After completion ofthe reaction, the reaction solution was passed through a Sephadex G-25column (46 mm ID×600 mm L, Pharmacia) equilibrated with 10% acetic acid.Elution with 10% acetic acid used for the equilibration at a flow rateof 6 mL/min gave the fraction containing the polypeptide (thepolypeptide having the amino acid sequence of SEQ ID NO: 7)/CS23 fusionprotein in the S-cyanized form. This effluent was concentrated anddesalted using the Pellicon Mini Cassette (Millipore) to obtain thepolypeptide (having the amino acid sequence of SEQ ID NO: 7)/CS23 fusionprotein in the desalted solution. After adding urea to this desaltedsolution at a final concentration of 6 M, 25% ammonia water was furtheradded at a final concentration of 3 M, and incubated for 15 min. at 15°C. for the reaction. After the reaction was completed, the solution wasadjusted to pH 6.0 with acetic acid to produce the polypeptide (havingthe amino acid sequence of SEQ ID NO: 7).

[0393] This reaction solution was passed through an SP-5PW column (5.5mm ID×30 cm L, Tosoh) for the absorption, which had been equilibratedwith 50 mM MES buffer solution containing 3 M urea (pH4.5). After thecolumn was washed, the elution was made with a gradient from 0 to 50% B(B=50 mM phosphate buffer solution+1 M NaCl+3 M urea) to obtain afraction containing the polypeptide (having the amino acid sequence ofSEQ ID NO: 7)(the elution time: 60 min). This polypeptide fraction wasfurther passed through an ODS-120T column (21.5 mm ID×300 mm L, Tosoh)for the absorption, which had been equilibrated with 0.1%trifluoroacetate (TFA). After the column was washed, the elution wasmade with a gradient from 30 to 60% B (B: 80% acetonitrile/0.1% TFA).The fractions containing the polypeptide of the invention (having theamino acid sequence of SEQ ID NO: 7)(the elution time: 45 min.) werepooled and lyophilized to obtain the polypeptide of the invention(having the amino acid sequence of SEQ ID NO: 7) as a lyophilizedpowder.

[0394] (a) Analysis of Amino Acid Composition

[0395] The amino acid composition was determined for the obtainedpolypeptide of the invention (the polypeptide having the amino acidsequence of SEQ ID NO: 7) using an amino acid analyzer (Hitachi L-8500A)(acid hydrolysis with 6N HCl-4% thioglycolic acid at 100° C. for 24, 48hrs).

[0396] The result was consistent with the amino acid compositionpredicted from the DNA sequence encoding the polypeptide of theinvention (the polypeptide having the amino acid sequence of SEQ ID NO:7) (Table 1). TABLE 1 Number predicted Number of residue from the DNAsequence of the Amino acid per mol present polypeptide Asx 4.2 5 Thr ¹⁾0.9 1 Ser ¹⁾ 2.7 3 Glx 2.0 2 Pro 3.9 4 Gly 1.0 1 Ala 1.9 2 Cys ²⁾ 0 Val1.7 2 Met 1.8 2 Ile 0.7 1 Leu 4 4 Tyr 0 0 Phe 2.8 3 His 1.0 1 Lys 3.7 4Arg 1.0 1 Trp 0.4 1

[0397] (b) Analysis of N-terminal Amino Acid Sequence

[0398] The N-terminal amino acid sequence was determined for theobtained polypeptide of the invention (the polypeptide having the aminoacid sequence of SEQ ID NO: 7) using a gas phase protein sequencer(Applied Biosystems, Model 477A)(100 pmol of the polypeptide of theinvention (the polypeptide having the amino acid sequence of SEQ ID NO:7) was used for analysis). The result show that the determinedN-terminal amino acid sequence was identical to the N-terminal aminoacid sequence predicted from the DNA sequence (Table 2). TABLE 2PTH¹⁾-amino acid detected Amino acid predicted from Residue No. (pmol)the base sequence of the present polypeptide 1 Ser (47) Ser 2 Leu (58)Leu 3 Asn (63) Asn 4 Phe (66) Phe 5 Glu (55) Glu 6 Glu (55) Glu 7 Leu(50) Leu 8 Lys (60) Lys 9 Asp (48) Asp 10 Trp (18) Trp 11 Gly (37) Gly12 Pro (26) Pro 13 Lys (48) Lys 14 Asn (27) Asn 15 Val (28) Val 16 Ile(25) Ile 17 Lys (46) Lys 18 Met (28) Met 19 Ser (12) Ser 20 Thr (11) Thr

[0399] (c) Analysis of C-terminal Amino Acid

[0400] The C-terminal amino acid was analyzed for the obtainedpolypeptide of the invention (the polypeptide having the amino acidsequence of SEQ ID NO: 7) using an amino acid analyzer (HitachiL-8500A), but there was no detection because the C-terninal wasamidated. TABLE 3 Analysis of C-terminal amino acid C-terminal aminoacid Recovery (%) RFRP-1 Phe —

Example 5

[0401] The activity of inhibiting the cAMP production was determined forthe polypeptide of the invention obtained in Example 4 (the polypeptidehaving the amino acid sequence of SEQ ID NO: 7) according to the methoddescribed in Example 1 of WO 00/29441. As a result, it was confirmedthat the said polypeptide has the activity as much as the syntheticpolypeptide (the polypeptide having the amino acid sequence of SEQ IDNO: 7).

INDUSTRIAL APPLICABILITY

[0402] Using the production method of the present invention, it ispossible to produce industrially in a large scale the polypeptide whichcan be used as a prophylactic or therapeutic agent for various diseasesassociated with hypo-secretion or hyper-secretion of prolactin.

1 30 1 9 PRT Human 1 Ser Phe Ala Asn Leu Pro Leu Arg Phe 1 5 9 2 27 DNAHuman 2 agctttgcga atctgccgct gcgtttt 27 3 12 PRT Human 3 Met Pro HisSer Phe Ala Asn Leu Pro Leu Arg Phe 1 5 10 12 4 36 DNA Human 4atgccgcata gctttgcgaa tctgccgctg cgtttt 36 5 20 PRT Human 5 Met Ser ThrPro Ala Val Asn Lys Met Pro His Ser Phe Ala Asn Leu 1 5 10 15 Pro LeuArg Phe 20 6 60 DNA Human 6 atgagcaccc cggcggtgaa taaaatgccg catagctttgcgaatctgcc gctgcgtttt 60 7 37 PRT Human 7 Ser Leu Asn Phe Glu Glu LeuLys Asp Trp Gly Pro Lys Asn Val Ile 1 5 10 15 Lys Met Ser Thr Pro AlaVal Asn Lys Met Pro His Ser Phe Ala Asn 20 25 30 Leu Pro Leu Arg Phe 3537 8 111 DNA Human 8 agcctgaact ttgaagaact gaaagattgg ggtccgaaaaatgtgattaa aatgagcacc 60 ccggcggtga ataaaatgcc gcatagcttt gcgaatctgccgctgcgttt t 111 9 8 PRT Human 9 Val Pro Asn Leu Pro Gln Arg Phe 1 5 810 24 DNA Human 10 gttcctaacc tgccccaaag gttt 24 11 17 PRT Human 11 AsnMet Glu Val Ser Leu Val Arg Arg Val Pro Asn Leu Pro Gln Arg 1 5 10 15Phe 17 12 51 DNA Human 12 aatatggagg tgagcctcgt gagacgtgtt cctaacctgccccaaaggtt t 51 13 146 PRT Human 13 Pro Ala Leu Pro Glu Asp Gly Gly SerGly Ala Phe Pro Pro Gly His 1 5 10 15 Phe Lys Asp Pro Lys Arg Leu TyrCys Lys Asn Gly Gly Phe Phe Leu 20 25 30 Arg Ile His Pro Asp Gly Arg ValAsp Gly Val Arg Glu Lys Ser Asp 35 40 45 Pro His Ile Lys Leu Gln Leu GlnAla Glu Glu Arg Gly Val Val Ser 50 55 60 Ile Lys Gly Val Ser Ala Asn ArgTyr Leu Ala Met Lys Glu Asp Gly 65 70 75 80 Arg Leu Leu Ala Ser Lys SerVal Thr Asp Glu Cys Phe Phe Phe Glu 85 90 95 Arg Leu Glu Ser Asn Asn TyrAsn Thr Tyr Arg Ser Arg Lys Tyr Thr 100 105 110 Ser Trp Tyr Val Ala LeuLys Arg Thr Gly Gln Tyr Lys Leu Gly Ser 115 120 125 Lys Thr Gly Pro GlyGln Lys Ala Ile Leu Phe Leu Pro Met Ser Ala 130 135 140 Lys Ser 145 14614 432 DNA Human 14 cccgaggatg gcggcagcgg cgccttcccg cccggccacttcaaggaccc caagcggctg 60 tactgcaaaa acgggggctt cttcctgcgc atccaccccgacggccgagt tgacggggtc 120 cgggagaaga gcgaccctca catcaagcta caacttcaagcagaagagag aggagttgtg 180 tctatcaaag gagtgagcgc taatcgttac ctggctatgaaggaagatgg aagattacta 240 gcttctaagt ctgttacgga tgagtgtttc ttttttgaacgattggaatc taataactac 300 aatacttacc ggtcaaggaa atacaccagt tggtatgtggcactgaaacg aactgggcag 360 tataaacttg gatccaaaac aggacctggg cagaaagctatactttttct tccaatgtct 420 gctaagagct gc 432 15 58 DNA ArtificialSequence Primer 15 tatgagcctg aactttgaag aactgaaaga ttggggtccgaaaaatgtga ttaaaatg 58 16 61 DNA Artificial Sequence Primer 16agcaccccgg cggtgaataa aatgccgcat agctttgcga atctgccgct gcgtttttgc 60 c61 17 62 DNA Artificial Sequence Primer 17 ggtgctcatt ttaatcacatttttcggacc ccaatctttc agttcttcaa agttcaggct 60 ca 62 18 59 DNAArtificial Sequence Primer 18 tcggggcaaa aacgcagcgg cagattcgcaaagctatgcg gcattttatt caccgccgg 59 19 180 PRT Human 19 Met Glu Ile IleSer Ser Lys Leu Phe Ile Leu Leu Thr Leu Ala Thr 1 5 10 15 Ser Ser LeuLeu Thr Ser Asn Ile Phe Cys Ala Asp Glu Leu Val Met 20 25 30 Ser Asn LeuHis Ser Lys Glu Asn Tyr Asp Lys Tyr Ser Glu Pro Arg 35 40 45 Gly Tyr ProLys Gly Glu Arg Ser Leu Asn Phe Glu Glu Leu Lys Asp 50 55 60 Trp Gly ProLys Asn Val Ile Lys Met Ser Thr Pro Ala Val Asn Lys 65 70 75 80 Met ProHis Ser Phe Ala Asn Leu Pro Leu Arg Phe Gly Arg Asn Val 85 90 95 Gln GluGlu Arg Ser Ala Gly Ala Thr Ala Asn Leu Pro Leu Arg Ser 100 105 110 GlyArg Asn Met Glu Val Ser Leu Val Arg Arg Val Pro Asn Leu Pro 115 120 125Gln Arg Phe Gly Arg Thr Thr Thr Ala Lys Ser Val Cys Arg Met Leu 130 135140 Ser Asp Leu Cys Gln Gly Ser Met His Ser Pro Cys Ala Asn Asp Leu 145150 155 160 Phe Tyr Ser Met Thr Cys Gln His Gln Glu Ile Gln Asn Pro AspGln 165 170 175 Lys Gln Ser Arg 180 20 540 DNA Human 20 atggaaattatttcatcaaa actattcatt ttattgactt tagccacttc aagcttgtta 60 acatcaaacattttttgtgc agatgaatta gtgatgtcca atcttcacag caaagaaaat 120 tatgacaaatattctgagcc tagaggatac ccaaaagggg aaagaagcct caattttgag 180 gaattaaaagattggggacc aaaaaatgtt attaagatga gtacacctgc agtcaataaa 240 atgccacactccttcgccaa cttgccattg agatttggga ggaacgttca agaagaaaga 300 agtgctggagcaacagccaa cctgcctctg agatctgga agaaatatgga ggtgagcctc 360 gtgagacgtgttcctaacct gccccaaagg tttgggagaa caacaacagc caaaagtgtc 420 tgcaggatgctgagtgattt gtgtcaagga tccatgcatt caccatgtgc caatgactta 480 ttttactccatgacctgcca gcaccaagaa atccagaatc ccgatcaaaa acagtcaagg 540 21 196 PRTHuman 21 Met Glu Ile Ile Ser Ser Lys Leu Phe Ile Leu Leu Thr Leu Ala Thr1 5 10 15 Ser Ser Leu Leu Thr Ser Asn Ile Phe Cys Ala Asp Glu Leu ValMet 20 25 30 Ser Asn Leu His Ser Lys Glu Asn Tyr Asp Lys Tyr Ser Glu ProArg 35 40 45 Gly Tyr Pro Lys Gly Glu Arg Ser Leu Asn Phe Glu Glu Leu LysAsp 50 55 60 Trp Gly Pro Lys Asn Val Ile Lys Met Ser Thr Pro Ala Val AsnLys 65 70 75 80 Met Pro His Ser Phe Ala Asn Leu Pro Leu Arg Phe Gly ArgAsn Val 85 90 95 Gln Glu Glu Arg Ser Ala Gly Ala Thr Ala Asn Leu Pro LeuArg Ser 100 105 110 Gly Arg Asn Met Glu Val Ser Leu Val Arg Arg Val ProAsn Leu Pro 115 120 125 Gln Arg Phe Gly Arg Thr Thr Thr Ala Lys Ser ValCys Arg Met Leu 130 135 140 Ser Asp Leu Cys Gln Gly Ser Met His Ser ProCys Ala Asn Asp Leu 145 150 155 160 Phe Tyr Ser Met Thr Cys Gln His GlnGlu Ile Gln Asn Pro Asp Gln 165 170 175 Lys Gln Ser Arg Arg Leu Leu PheLys Lys Ile Asp Asp Ala Glu Leu 180 185 190 Lys Gln Glu Lys 195 22 588DNA Human 22 atggaaatta tttcatcaaa actattcatt ttattgactt tagccacttcaagcttgtta 60 acatcaaaca ttttttgtgc agatgaatta gtgatgtcca atcttcacagcaaagaaaat 120 tatgacaaat attctgagcc tagaggatac ccaaaagggg aaagaagcctcaattttgag 180 gaattaaaag attggggacc aaaaaatgtt attaagatga gtacacctgcagtcaataaa 240 atgccacact ccttcgccaa cttgccattg agatttggga ggaacgttcaagaagaaaga 300 agtgctggag caacagccaa cctgcctctg agatctggaa gaaatatggaggtgagcctc 360 gtgagacgtg ttcctaacct gccccaaagg tttgggagaa caacaacagccaaaagtgtc 420 tgcaggatgc tgagtgattt gtgtcaagga tccatgcatt caccatgtgccaatgactta 480 ttttactcca tgacctgcca gcaccaagaa atccagaatc ccgatcaaaaacagtcaagg 540 agactgctat tcaagaaaat agatgatgca gaattgaaac aagaaaaa 58823 196 PRT Bovine 23 Met Glu Ile Ile Ser Leu Lys Arg Phe Ile Leu Leu MetLeu Ala Thr 1 5 10 15 Ser Ser Leu Leu Thr Ser Asn Ile Phe Cys Thr AspGlu Ser Arg Met 20 25 30 Pro Asn Leu Tyr Ser Lys Lys Asn Tyr Asp Lys TyrSer Glu Pro Arg 35 40 45 Gly Asp Leu Gly Trp Glu Lys Glu Arg Ser Leu ThrPhe Glu Glu Val 50 55 60 Lys Asp Trp Ala Pro Lys Ile Lys Met Asn Lys ProVal Val Asn Lys 65 70 75 80 Met Pro Pro Ser Ala Ala Asn Leu Pro Leu ArgPhe Gly Arg Asn Met 85 90 95 Glu Glu Glu Arg Ser Thr Arg Ala Met Ala HisLeu Pro Leu Arg Leu 100 105 110 Gly Lys Asn Arg Glu Asp Ser Leu Ser ArgTrp Val Pro Asn Leu Pro 115 120 125 Gln Arg Phe Gly Arg Thr Thr Thr AlaLys Ser Ile Thr Lys Thr Leu 130 135 140 Ser Asn Leu Leu Gln Gln Ser MetHis Ser Pro Ser Thr Asn Gly Leu 145 150 155 160 Leu Tyr Ser Met Ala CysGln Pro Gln Glu Ile Gln Asn Pro Gly Gln 165 170 175 Lys Asn Leu Arg ArgArg Gly Phe Gln Lys Ile Asp Asp Ala Glu Leu 180 185 190 Lys Gln Glu Lys195 24 588 DNA Bovine 24 atggaaatta tttcattaaa acgattcatt ttattgatgttagccacttc aagcttgtta 60 acatcaaaca tcttctgcac agacgaatca aggatgcccaatctttacag caaaaagaat 120 tatgacaaat attccgagcc tagaggagat ctaggctgggagaaagaaag aagtcttact 180 tttgaagaag taaaagattg ggctccaaaa attaagatgaataaacctgt agtcaacaaa 240 atgccacctt ctgcagccaa cctgccactg agatttgggaggaacatgga agaagaaagg 300 agcactaggg cgatggccca cctgcctctg agactcggaaaaaatagaga ggacagcctc 360 tccagatggg tcccaaatct gccccagagg tttggaagaacaacaacagc caaaagcatt 420 accaagaccc tgagtaattt gctccagcag tccatgcattcaccatctac caatgggcta 480 ctctactcca tggcctgcca gccccaagaa atccagaatcctggtcaaaa gaacctaagg 540 agacggggat tccagaaaat agatgatgca gaattgaaacaagaaaaa 588 25 188 PRT Mouse 25 Met Glu Ile Ile Ser Leu Lys Arg Phe IleLeu Leu Thr Val Ala Thr 1 5 10 15 Ser Ser Phe Leu Thr Ser Asn Thr PheCys Thr Asp Glu Phe Met Met 20 25 30 Pro His Phe His Ser Lys Glu Gly AspGly Lys Tyr Ser Gln Leu Arg 35 40 45 Gly Ile Pro Lys Gly Glu Lys Glu ArgSer Val Ser Phe Gln Glu Leu 50 55 60 Lys Asp Trp Gly Ala Lys Asn Val IleLys Met Ser Pro Ala Pro Ala 65 70 75 80 Asn Lys Val Pro His Ser Ala AlaAsn Leu Pro Leu Arg Phe Gly Arg 85 90 95 Thr Ile Asp Glu Lys Arg Ser ProAla Ala Arg Val Asn Met Glu Ala 100 105 110 Gly Thr Arg Ser His Phe ProSer Leu Pro Gln Arg Phe Gly Arg Thr 115 120 125 Thr Ala Arg Ser Pro LysThr Pro Ala Asp Leu Pro Gln Lys Pro Leu 130 135 140 His Ser Leu Gly SerSer Glu Leu Leu Tyr Val Met Ile Cys Gln His 145 150 155 160 Gln Glu IleGln Ser Pro Gly Gly Lys Arg Thr Arg Arg Gly Ala Phe 165 170 175 Val GluThr Asp Asp Ala Glu Arg Lys Pro Glu Lys 180 185 26 564 DNA Mouse 26atggaaatta tttcattaaa acgattcatt ttattgactg tggcaacttc aagcttctta 60acatcaaaca ccttctgtac agatgagttc atgatgcctc attttcacag caaagaaggt 120gacggaaaat actcccagct gagaggaatc ccaaaagggg aaaaggaaag aagtgtcagt 180tttcaagaac taaaagattg gggggcaaag aatgttatta agatgagtcc agcccctgcc 240aacaaagtgc cccactcagc agccaacctg cccctgagat ttggaaggac catagatgag 300aaaagaagcc ccgcagcacg ggtcaacatg gaggcaggga ccaggagcca tttccccagc 360ctgccccaaa ggtttgggag aacaacagcc agaagcccca agacacccgc tgatttgcca 420cagaaacccc tgcactcact gggctccagc gagttgctct acgtcatgat ctgccagcac 480caagaaattc agagtcctgg tggaaagcga acgaggagag gagcgtttgt ggaaacagat 540gatgcagaaa ggaaaccaga aaaa 564 27 203 PRT Rat 27 Met Glu Ile Ile Ser SerLys Arg Phe Ile Leu Leu Thr Leu Ala Thr 1 5 10 15 Ser Ser Phe Leu ThrSer Asn Thr Leu Cys Ser Asp Glu Leu Met Met 20 25 30 Pro His Phe His SerLys Glu Gly Tyr Gly Lys Tyr Tyr Gln Leu Arg 35 40 45 Gly Ile Pro Lys GlyVal Lys Glu Arg Ser Val Thr Phe Gln Glu Leu 50 55 60 Lys Asp Trp Gly AlaLys Lys Asp Ile Lys Met Ser Pro Ala Pro Ala 65 70 75 80 Asn Lys Val ProHis Ser Ala Ala Asn Leu Pro Leu Arg Phe Gly Arg 85 90 95 Asn Ile Glu AspArg Arg Ser Pro Arg Ala Arg Ala Asn Met Glu Ala 100 105 110 Gly Thr MetSer His Phe Pro Ser Leu Pro Gln Arg Phe Gly Arg Thr 115 120 125 Thr AlaArg Arg Ile Thr Lys Thr Leu Ala Gly Leu Pro Gln Lys Ser 130 135 140 LeuHis Ser Leu Ala Ser Ser Glu Leu Leu Tyr Ala Met Thr Arg Gln 145 150 155160 His Gln Glu Ile Gln Ser Pro Gly Gln Glu Gln Pro Arg Lys Arg Val 165170 175 Phe Thr Glu Thr Asp Asp Ala Glu Arg Lys Gln Glu Lys Ile Gly Asn180 185 190 Leu Gln Pro Val Leu Gln Gly Ala Met Lys Leu 195 200 28 609DNA Rat 28 atggaaatta tttcatcaaa gcgattcatt ttattgactt tagcaacttcaagcttctta 60 acttcaaaca ccctttgttc agatgaatta atgatgcccc attttcacagcaaagaaggt 120 tatggaaaat attaccagct gagaggaatc ccaaaagggg taaaggaaagaagtgtcact 180 tttcaagaac tcaaagattg gggggcaaag aaagatatta agatgagtccagcccctgcc 240 aacaaagtgc cccactcagc agccaacctt cccctgaggt ttgggaggaacatagaagac 300 agaagaagcc ccagggcacg ggccaacatg gaggcaggga ccatgagccattttcccagc 360 ctgccccaaa ggtttgggag aacaacagcc agacgcatca ccaagacactggctggtttg 420 ccccagaaat ccctgcactc cctggcctcc agtgaattgc tctatgccatgacccgccag 480 catcaagaaa ttcagagtcc tggtcaagag caacctagga aacgggtgttcacggaaaca 540 gatgatgcag aaaggaaaca agaaaaaata ggaaacctcc agccagtccttcaaggggct 600 atgaagctg 609 29 203 PRT Rat 29 Met Glu Ile Ile Ser SerLys Arg Phe Ile Leu Leu Thr Leu Ala Thr 1 5 10 15 Ser Ser Phe Leu ThrSer Asn Thr Leu Cys Ser Asp Glu Leu Met Met 20 25 30 Pro His Phe His SerLys Glu Gly Tyr Gly Lys Tyr Tyr Gln Leu Arg 35 40 45 Gly Ile Pro Lys GlyVal Lys Glu Arg Ser Val Thr Phe Gln Glu Leu 50 55 60 Lys Asp Trp Gly AlaLys Lys Asp Ile Lys Met Ser Pro Ala Pro Ala 65 70 75 80 Asn Lys Val ProHis Ser Ala Ala Asn Leu Pro Leu Arg Phe Gly Arg 85 90 95 Asn Ile Glu AspArg Arg Ser Pro Arg Ala Arg Ala Asn Met Glu Ala 100 105 110 Gly Thr MetSer His Phe Pro Ser Leu Pro Gln Arg Phe Gly Arg Thr 115 120 125 Thr AlaArg Arg Ile Thr Lys Thr Leu Ala Gly Leu Pro Gln Lys Ser 130 135 140 LeuHis Ser Leu Ala Ser Ser Glu Ser Leu Tyr Ala Met Thr Arg Gln 145 150 155160 His Gln Glu Ile Gln Ser Pro Gly Gln Glu Gln Pro Arg Lys Arg Val 165170 175 Phe Thr Glu Thr Asp Asp Ala Glu Arg Lys Gln Glu Lys Ile Gly Asn180 185 190 Leu Gln Pro Val Leu Gln Gly Ala Met Lys Leu 195 200 30 609DNA Rat 30 atggaaatta tttcatcaaa gcgattcatt ttattgactt tagcaacttcaagcttctta 60 acttcaaaca ccctttgttc agatgaatta atgatgcccc attttcacagcaaagaaggt 120 tatggaaaat attaccagct gagaggaatc ccaaaagggg taaaggaaagaagtgtcact 180 tttcaagaac tcaaagattg gggggcaaag aaagatatta agatgagtccagcccctgcc 240 aacaaagtgc cccactcagc agccaacctt cccctgaggt ttgggaggaacatagaagac 300 agaagaagcc ccagggcacg ggccaacatg gaggcaggga ccatgagccattttcccagc 360 ctgccccaaa ggtttgggag aacaacagcc agacgcatca ccaagacactggctggtttg 420 ccccagaaat ccctgcactc cctggcctcc agtgaatcgc tctatgccatgacccgccag 480 catcaagaaa ttcagagtcc tggtcaagag caacctagga aacgggtgttcacggaaaca 540 gatgatgcag aaaggaaaca agaaaaaata ggaaacctcc agccagtccttcaaggggct 600 atgaagctg 609

1. A method of producing a partial peptide of the polypeptide comprisingthe amino acid sequence of SEQ ID NO: 19, 21, 23, 25, 27 or 29, or asalt thereof, which comprises: subjecting a fusion protein orpolypeptide, in which a partial peptide of the polypeptide comprisingthe amino acid sequence of SEQ ID NO: 19, 21, 23, 25, 27 or 29, whichmay has a methionine residue optionally oxidized at the N-terminal, isligated to the N-terninal of a protein or polypeptide having a cysteineat the N-terminal, to a reaction for cleavage of a peptide bond on theamino group side of the cysteine residue.
 2. A method of producing apartial peptide of the polypeptide comprising the amino acid sequence ofSEQ ID NO: 19, 21, 23, 25, 27 or 29, which may has a methionine residueat the N-terminal, or a salt thereof, which comprises: culturing atransformant containing a vector having a DNA encoding a fusion proteinor polypeptide, in which the partial peptide of the polypeptidecomprising the amino acid sequence of SEQ ID NO: 19, 21, 23, 25, 27 or29, which may has a methionine residue at the N-terminal, is ligated tothe N-terminal of a protein or polypeptide having a cysteine at theN-terminal to express the fusion protein or polypeptide; and subjectingthe expressed fusion protein or polypeptide to a reaction for cleavageof a peptide bond on the amino group side of the cysteine residue. 3.The production method of claim 1 or 2, wherein the cleavage reaction isS-cyaniation reaction followed by an ammonolysis or hydrolysis reaction.4. The production method of claim 1 or 2, wherein the partial peptide ofthe polypeptide comprising the amino acid sequence of SEQ ID NO: 19, 21,23, 25, 27 or 29 includes: (i) a peptide comprising the amino acidsequence from aa 56 (Ser) to aa 92 (Phe), aa 73 (Met) to aa 92 (Phe), aa81 (Met) to aa 92(Phe), aa 84 (Ser) to aa 92 (Phe), aa 101 (Ser) to aa112 (Ser), aa 95 (Asn) to aa 112 (Ser), aa 115 (Asn) to aa 131 (Phe), aa124 (Val) to aa 131 (Phe), aa 1 (Met) to aa 92 (Phe), aa 1 (Met) to aa112 (Ser), or aa 1 (Met) to aa 131 (Phe) in the amino acid sequence ofSEQ ID NO: 19; (ii) a peptide comprising the amino acid sequence from aa56 (Ser) to aa 92 (Phe), aa 73 (Met) to aa 92 (Phe), aa 81 (Met) to aa92 (Phe), aa 84 (Ser) to aa 92 (Phe), aa 101 (Ser) to aa 112 (Ser), aa95 (Asn) to aa 112 (Ser), aa 115 (Asn) to aa 131 (Phe), aa 124 (Val) toaa 131 (Phe), aa 1 (Met) to aa 92 (Phe), aa 1 (Met) to aa 112 (Ser) oraa 1 (Met) to aa 131 (Phe) in the amino acid sequence of SEQ ID NO: 21;(iii) a peptide comprising the amino acid sequence from aa 58 (Ser) toaa 92 (Phe), aa 81 (Met) to aa 92 (Phe), aa 101 (Ser) to aa 112 (Leu),aa 95 (Asn) to aa 112 (Leu), aa 124 (Val) to aa 131 (Phe), aa 1 (Met) toaa 92 (Phe), or aa 1 (Met) to aa 131 (Phe) in the amino acid sequence ofSEQ ID NO: 23; (iv) a peptide comprising the amino acid sequence from aa58 (Ser) to aa 94 (Phe), aa 83 (Val) to aa 94 (Phe), aa 84 (Pro) to aa94 (Phe), or aa 118 (Phe) to aa 125 (Phe) in the amino acid sequence ofSEQ ID NO: 25; or (v) a peptide comprising the amino acid sequence fromaa 58 (Ser) to aa 94 (Phe), or aa 84 (Pro) to aa 94 (Phe) in the aminoacid sequence of SEQ ID NO:
 27. 5. The production method of claim 1 or2, wherein the partial peptide of the polypeptide comprising the aminoacid sequence of SEQ ID NO: 19, 21, 23, 25, 27 or 29 includes thepolypeptide comprising the amino acid sequence of SEQ ID NO: 1 or
 9. 6.The production method of claim 5, wherein the polypeptide comprising theamino acid sequence of SEQ ID NO: 1 or 9 includes the polypeptide havingthe amino acid sequence of,SEQ ID NO: 1, 3, 5, 7, 9 or
 11. 7. A fusionprotein or polypeptide, in which a partial peptide of the polypeptidecomprising the amino acid sequence of SEQ ID NO: 19, 21, 23, 25, 27 or29, which may has a methionine residue at the N-terminal, is ligated tothe N-terminal of a protein or polypeptide having a cysteine at theN-terminal, or a salt thereof.
 8. The polypeptide of claim 7 or a saltthereof, wherein the partial peptide of the polypeptide comprising theamino acid sequence of SEQ ID NO: 19, 21, 23, 25, 27 or 29 includes: (i)a peptide comprising the amino acid sequence from aa 56 (Ser) to aa 92(Phe), aa 73 (Met) to aa 92 (Phe), aa 81 (Met) to aa 92(Phe), aa 84(Ser) to aa 92 (Phe), aa 101 (Ser) to aa 112 (Ser), aa 95 (Asn) to aa112 (Ser), aa 115 (Asn) to aa 131 (Phe), aa 124 (Val) to aa 131 (Phe),aa 1 (Met) to aa 92 (Phe), aa 1 (Met) to aa 112 (Ser), or aa 1 (Met) toaa 131 (Phe) in the amino acid sequence of SEQ ID NO: 19; (ii) a peptidecomprising the amino acid sequence from aa 56 (Ser) to aa 92 (Phe), aa73 (Met) to aa 92 (Phe), aa 81 (Met) to aa 92 (Phe), aa 84 (Ser) to aa92 (Phe), aa 101 (Ser) to aa 112 (Ser), aa 95 (Asn) to aa 112 (Ser), aa115 (Asn) to aa 131 (Phe), aa 124 (Val) to aa 131 (Phe), aa 1 (Met) toaa 92 (Phe), aa 1 (Met) to aa 112 (Ser) or aa 1 (Met) to aa 131 (Phe) inthe amino acid sequence of SEQ ID NO: 21; (iii) a peptide comprising theamino acid sequence from aa 58 (Ser) to aa 92 (Phe), aa 81 (Met) to aa92 (Phe), aa 101 (Ser) to aa 112 (Leu), aa 95 (Asn) to aa 112 (Leu), aa124 (Val) to aa 131 (Phe), aa 1 (Met) to aa 92 (Phe), or aa 1 (Met) toaa 131 (Phe) in the amino acid sequence of SEQ ID NO: 23; (iv) a peptidecomprising the amino acid sequence from aa 58 (Ser) to aa 94 (Phe), aa83 (Val) to aa 94 (Phe), aa 84 (Pro) to aa 94 (Phe), or aa 118 (Phe) toaa 125 (Phe) in the amino acid sequence of SEQ ID NO: 25; or (v) apeptide comprising the amino acid sequence from aa 58 (Ser) to aa 94(Phe), or aa 84 (Pro) to aa 94 (Phe) in the amino acid sequence of SEQID NO:
 27. 9. The polypeptide of claim 7 or a salt thereof, wherein thepartial peptide of the polypeptide comprising the amino acid sequence ofSEQ ID NO: 19, 21, 23, 25, 27 or 29 includes the polypeptide comprisingthe amino acid sequence of SEQ ID NO: 1 or
 9. 10. The polypeptide ofclaim 9 or a salt thereof, wherein the polypeptide comprising the aminoacid sequence of SEQ ID NO: 1 or 9 includes the polypeptide having theamino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9 or
 11. 11. A vectorcomprising the DNA encoding the fusion protein or polypeptide of claim7.
 12. A transfornant having the vector of claim
 11. 13. A method ofremoving the N-terminal methionine residue from a partial peptide of thepolypeptide comprising the amino acid sequence of SEQ ID NO: 19, 21, 23,25, 27 or 29, which has a methionine residue optionally oxidized at theN-terminal, which comprises: reacting the partial peptide or a saltthereof with α-diketone; and then subjecting it to a hydrolysisreaction.
 14. The method of claim 13, wherein the partial peptide of thepolypeptide comprising the amino acid sequence of SEQ ID NO: 19, 21, 23,25, 27 or 29, which has a methionine residue optionally oxidized at theN-terminal, is produced by genetic engineering, and the amino acid nextto the methionine residue on the C-terminal side is Isoleucine, Valine,Cysteine, Threonine, Aspartic acid, Lysine, Leucine, Arginine,Asparagine, Metbionine, Phenylalanine, Tyrosine, Tryptophan, Glutamicacid, Glutamine or Histidine.
 15. The method of claim 13 or 14, whereinthe partial peptide of the polypeptide comprising the amino acidsequence of SEQ ID NO: 19, 21, 23, 25, 27 or 29 includes: (i) a peptidecomprising the amino acid sequence from aa 56 (Ser) to aa 92 (Phe), aa73 (Met) to aa 92 (Phe), aa 81 (Met) to aa 92(Phe), aa 84 (Ser) to aa 92(Phe), aa 101 (Ser) to aa 112 (Ser), aa 95 (Asn) to aa 112 (Ser), aa 115(Asn) to aa 131 (Phe), aa 124 (Val) to aa 131 (Phe), aa 1 (Met) to aa 92(Phe), aa 1 (Met) to aa 112 (Ser), or aa 1 (Met) to aa 131 (Phe) in theamino acid sequence of SEQ ID NO: 19; (ii) a peptide comprising theamino acid sequence from aa 56 (Ser) to aa 92 (Phe), aa 73 (Met) to aa92 (Phe), aa 81 (Met) to aa 92 (Phe), aa 84 (Ser) to aa 92 (Phe), aa 101(Ser) to aa 112 (Ser), aa 95 (Asn) to aa 112 (Ser), aa 115 (Asn) to aa131 (Phe), aa 124 (Val) to aa 131 (Phe), aa 1 (Met) to aa 92 (Phe), aa 1(Met) to aa 112 (Ser) or aa 1 (Met) to aa 131 (Phe) in the amino acidsequence of SEQ ID NO: 21; (iii) a peptide comprising the amino acidsequence from aa 58 (Ser) to aa 92 (Phe), aa 81 (Met) to aa 92 (Phe), aa101 (Ser) to aa 112 (Leu), aa 95 (Asn) to aa 112 (Leu), aa 124 (Val) toaa 131 (Phe), aa 1 (Met) to aa 92 (Phe), or aa 1 (Met) to aa 131 (Phe)in the amino acid sequence of SEQ ID NO: 23; (iv) a peptide comprisingthe amino acid sequence from aa 58 (Ser) to aa 94 (Phe), aa 83 (Val) toaa 94 (Phe), aa 84 (Pro) to aa 94 (Phe), or aa 118 (Phe) to aa 125 (Phe)in the amino acid sequence of SEQ ID NO: 25; or (v) a peptide comprisingthe amino acid sequence from aa 58 (Ser) to aa 94 (Phe), or aa 84 (Pro)to aa 94 (Phe) in the amino acid sequence of SEQ ID NO:
 27. 16. Themethod of claim 13 or 14, wherein the partial peptide of the polypeptidecomprising the amino acid sequence of SEQ ID NO: 19, 21, 23, 25, 27 or29 includes the polypeptide comprising the amino acid sequence of SEQ IDNO: 1 or
 9. 17. The method of claim 16, wherein the polypeptidecomprising the amino acid sequence of SEQ ID NO: 1 or 9 includes thepolypeptide having the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9or
 11. 18. The method of claim 13, wherein the reaction with α-diketoneis carried out in the presence of a transition metal ion.
 19. The methodof claim 13, wherein the reaction with α-diketone is carried out in thepresence of a base.
 20. The method of claim 13, wherein the reactionwith α-diketone is carried out in the presence of a transition metal ionand a base.
 21. The method of claim 13, wherein the α-diketone isglyoxylic acid or its salt.
 22. The method of claim 18, wherein thetransition metal ion is a copper ion.
 23. The method of claim 19,wherein the base is pyridine.
 24. The method of claim 13, wherein thehydrolysis reaction is carried out using a base.
 25. The method of claim24, wherein the base is an amine.
 26. The method of claim 24, whereinthe base is a diamine, or thio- or seleno-semicarbazide.
 27. The methodof claim 26, wherein the diamine is o-phenylenediamine or3,4-diamino-benzoic acid.
 28. A method of producing the polypeptidecomprising the amino acid sequence of SEQ ID NO: 1 or 9, or a saltthereof, which comprises: reacting a partial peptide of the polypeptidecomprising the amino acid sequence of SEQ ID NO: 19, 21, 23, 25, 27 or29, which has a methionine residue added at the N-terminal, or a saltthereof, which is produced by genetic engineering, with glyoxylic acidor its salt in the presence of copper sulfate and pyridine; and thenreacting it with o-phenylenediamine or 3,4-diamino-benzoic acid.
 29. Theproduction method of claim 28, wherein the partial peptide of thepolypeptide comprising the amino acid sequence of SEQ ID NO: 19, 21, 23,25, 27 or 29 includes: (i) a peptide comprising the amino acid sequencefrom aa 56 (Ser) to aa 92 (Phe), aa 73 (Met) to aa 92 (Phe), aa 81 (Met)to aa 92(Phe), aa 84 (Ser) to aa 92 (Phe), aa 101 (Ser) to aa 112 (Ser),aa 95 (Asn) to aa 112 (Ser), aa 115 (Asn) to aa 131 (Phe), aa 124 (Val)to aa 131 (Phe), aa 1 (Met) to aa 92 (Phe), aa 1 (Met) to aa 112 (Ser),or aa 1 (Met) to aa 131 (Phe) in the amino acid sequence of SEQ ID NO:19; (ii) a peptide comprising the amino acid sequence from aa 56 (Ser)to aa 92 (Phe), aa 73 (Met) to aa 92 (Phe), aa 81 (Met) to aa 92 (Phe),aa 84 (Ser) to aa 92 (Phe), aa 101 (Ser) to aa 112 (Ser), aa 95 (Asn) toaa 112 (Ser), aa 115 (Asn) to aa 131 (Phe), aa 124 (Val) to aa 131(Phe), aa 1 (Met) to aa 92 (Phe), aa 1 (Met) to aa 112 (Ser) or aa 1(Met) to aa 131 (Phe) in the amino acid sequence of SEQ ID NO: 21; (iii)a peptide comprising the amino acid sequence from aa 58 (Ser) to aa 92(Phe), aa 81 (Met) to aa 92 (Phe), aa 101 (Ser) to aa 112 (Leu), aa 95(Asn) to aa 112 (Leu), aa 124 (Val) to aa 131 (Phe), aa 1 (Met) to aa 92(Phe), or aa 1 (Met) to aa 131 (Phe) in the amino acid sequence of SEQID NO: 23; (iv) a peptide comprising the amino acid sequence from aa 58(Ser) to aa 94 (Phe), aa 83 (Val) to aa 94 (Phe), aa 84 (Pro) to aa 94(Phe), or aa 118 (Phe) to aa 125 (Phe) in the amino acid sequence of SEQID NO: 25; or (v) a peptide comprising the amino acid sequence from aa58 (Ser) to aa 94 (Phe), or aa 84 (Pro) to aa 94 (Phe) in the amino acidsequence of SEQ ID NO:
 27. 30. The production method of claim 28,wherein the partial peptide of the polypeptide comp rising the aminoacid sequence of SEQ ID NO: 19, 21, 23, 25, 27 or 29 includes thepolypeptide comprising the amino acid sequence of SEQ ID NO: 1 or
 9. 31.The production method of claim 30, wherein the polypeptide comprisingthe amino acid sequence of SEQ ID NO: 1 or 9 includes the polypeptidehaving the amino acid sequence of SEQ ID NO: I, 3, 5, 7, 9 or
 11. 32. Acompound represented by the formula CH₃—S(O)_(m)—(CH₂)₂—CO—CO—X, or asalt thereof, wherein m indicates an integer of 0 to 2, and X indicatesa partial peptide chain of the polypeptide comprising the amino acidsequence of SEQ ID NO: 19, 21, 23, 25, 27 or
 29. 33. The compound ofclaim 32, or a salt thereof, wherein the partial peptide of thepolypeptide comprising the amino acid sequence of SEQ ID NO: 19, 21, 23,25, 27 or 29 includes: (i) a peptide comprising the amino acid sequencefrom aa 56 (Ser) to aa 92 (Phe), aa 73 (Met) to aa 92 (Phe), aa 81 (Met)to aa 92(Phe), aa 84 (Ser) to aa 92 (Phe), aa 101 (Ser) to aa 112 (Ser),aa 95 (Asn) to aa 112 (Ser), aa 115 (Asn) to aa 131 (Phe), aa 124 (Val)to aa 131 (Phe), aa 1 (Met) to aa 92 (Phe), aa 1 (Met) to aa 112 (Ser),or aa 1 (Met) to aa 131 (Phe) in the amino acid sequence of SEQ ID NO:19; (ii) a peptide comprising the amino acid sequence from aa 56 (Ser)to aa 92 (Phe), aa 73 (Met) to aa 92 (Phe), aa 81 (Met) to aa 92 (Phe),aa 84 (Ser) to aa 92 (Phe), aa 101 (Ser) to aa 112 (Ser), aa 95 (Asn) toaa 112 (Ser), aa 115 (Asn) to aa 131 (Phe), aa 124 (Val) to aa 131(Phe), aa 1 (Met) to aa 92 (Phe), aa 1 (Met) to aa 112 (Ser) or aa 1(Met) to aa 131 (Phe) in the amino acid sequence of SEQ ID NO: 21; (iii)a peptide comprising the amino acid sequence from aa 58 (Ser) to aa 92(Phe), aa 81 (Met) to aa 92 (Phe), aa 101 (Ser) to aa 112 (Leu), aa 95(Asn) to aa 112 (Leu), aa 124 (Val) to aa 131 (Phe), aa 1 (Met) to aa 92(Phe), or aa 1 (Met) to aa 131 (Phe) in the amino acid sequence of SEQID NO: 23; (iv) a peptide comprising the amino acid sequence from aa 58(Ser) to aa 94 (Phe), aa 83 (Val) to aa 94 (Phe), aa 84 (Pro) to aa 94(Phe), or aa 118 (Phe) to aa 125 (Phe) in the amino acid sequence of SEQID NO: 25; or (v) a peptide comprising the amino acid sequence from aa58 (Ser) to aa 94 (Phe), or aa 84 (Pro) to aa 94 (Phe) in the amino acidsequence of SEQ ID NO:
 27. 34. The compound of claim 32, or a saltthereof, wherein the partial peptide of the polypeptide comprising theamino acid sequence of SEQ ID NO: 19, 21, 23, 25, 27 or 29 includes thepolypeptide comprising the amino acid sequence of SEQ ID NO: 1 or
 9. 35.The compound of claim 34, or a salt thereof, wherein the polypeptidecomprising the amino acid sequence of SEQ ID NO: 1 or 9 includes thepolypeptide having the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9or
 11. 36. A method of producing a partial peptide of the polypeptidecomprising the amino acid sequence of SEQ ID NO: 19, 21, 23, 25, 27 or29, or a salt thereof, which comprises subjecting the compound of claim32 to a hydrolysis reaction.
 37. A method of producing the polypeptidecomprising the amino acid sequence represented by, or a salt thereof,which comprises: subjecting a fusion protein or polypeptide, in whichthe polypeptide comprising the amino acid sequence represented by, whichhas a methionine residue at the N-terminal, is ligated to the N-terminalof a protein or polypeptide having a cysteine at the N-terminal, to areaction for cleavage of a peptide bond on the amino group side of thecysteine residue to obtain the polypeptide comprising the amino acidsequence represented by, which has a methionine residue optionallyoxidized at the N-terminal, or a salt thereof; reacting the polypeptidecomprising the amino acid sequence represented by, which has amethioninie residue optionally oxidized at the N-terminal, or a saltthereof with α-diketone; and subjecting it to a hydrolysis reaction. 38.The production method of claim 37, wherein the partial peptide of thepolypeptide comprising the amino acid sequence of SEQ ID NO: 19, 21, 23,25, 27 or 29 includes: (i) a peptide comprising the amino acid sequencefrom aa 56 (Ser) to aa 92 (Phe), aa 73 (Met) to aa 92 (Phe), aa 81 (Met)to aa 92(Phe), aa 84 (Ser) to aa 92 (Phe), aa 101 (Ser) to aa 112 (Ser),aa 95 (Asn) to aa 112 (Ser), aa 115 (Asn) to aa 131 (Phe), aa 124 (Val)to aa 131 (PIle), aa 1 (Met) to aa 92 (Phe), aa 1 (Met) to aa 112 (Ser),or aa 1 (Met) to aa 131 (Phe) in the amino acid sequence of SEQ ID NO:19; (ii) a peptide comprising the amino acid sequence from aa 56 (Ser)to aa 92 (Phe), aa 73 (Met) to aa 92 (Phe), aa 81 (Met) to aa 92 (Phe),aa 84 (Ser) to aa 92 (Phe), aa 101 (Ser) to aa 112 (Ser), aa 95 (Asn) toaa 112 (Ser), aa 115 (Asn) to aa 131 (Phe), aa 124 (Val) to aa 131(Phe), aa 1 (Met) to aa 92 (Phe), aa 1 (Met) to aa 112 (Ser) or aa 1(Met) to aa 131 (Phe) in the amino acid sequence of SEQ ID NO: 21; (iii)a peptide comprising the amino acid sequence from aa 58 (Ser) to aa 92(Phe), aa 81 (Met) to aa 92 (Phe), aa 101 (Ser) to aa 112 (Leu), aa 95(Asn) to aa 112 (Leu), aa 124 (Val) to aa 131 (Phe), aa 1 (Met) to aa 92(Phe), or aa 1 (Met) to aa 131 (Phe) in the amino acid sequence of SEQID NO: 23; (iv) a peptide comprising the amino acid sequence from aa 58(Ser) to aa 94 (Phe), aa 83 (Val) to aa 94 (Phe), aa 84 (Pro) to aa 94(Phe), or aa 118 (Phe) to aa 125 (Phe) in the amino acid sequence of SEQID NO: 25; or (v) a peptide comprising the amino acid sequence from aa58 (Ser) to aa 94 (Phe), or aa 84 (Pro) to aa 94 (Phe) in the amino acidsequence of SEQ ID NO:
 27. 39. The production method of claim 37,wherein the partial peptide of the polypeptide comprising the amino acidsequence of SEQ ID NO: 19, 21, 23, 25, 27 or 29 includes the polypeptidecomprising the amino acid sequence of SEQ ID NO: 1 or
 9. 40. Theproduction method of claim 39, wherein the polypeptide comprising theamino acid sequence of SEQ ID NO: 1 or 9 includes the polypeptide havingthe amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9 or 11.